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PCT

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :
C12N 9/20, 9/18, CUD 3/386

(11) International Publication Number; WO 97/04079

(43) International Publication Date: 6 February 1997 (06.02.97)

(21) International Application Number: PCI7DK96700322

(22) International FQing Date: 12 July 1996 (12.07.96)

(30) Priority Data:

0832/95

14 July 1995 (14.07.95)

1013/95

13 September 1995 (13.09.95)

1096/95

29 September 1995 (29.09.95)

1306795

21 November 1995 (21.11.95)

60/011,634

14 February 1996 (14.0X96)

0372/96

1 April 1996(01.04.96)

60/020.461

7 May 1996 (07.05.96)

(71) Applicant {for all designated States except US): NOVO

NORDISK A/S [DK/DK]; Novo Alfc, DK-2880 Bagsvsrd
(DK).

(72) Inventors; and

(75) Inventors/Applicants (for US only): FUGLSANG, Clans,
Crone [DK/DK); Novo Nordisk a/s. Novo Alle\ DK-
2880 Bagsvasrd (DK). OKKELS, Jens, Sigurd [DK/DK];
Novo Nordisk a/s, Novo AI16, DK-2880 Bagsvserd (DK).
PETERSEN, Dorte, Aaby [DK/DK]; Novo Nordisk a/s.
Novo Alle*, DK-2880 Bagsvaerd (DK). PATKAR, Shamkant,
Anant [DK/DK]; Novo Nordisk a/s. Novo AIle\ DK-
2880 Bagsvaerd (DK). THELLERSEN, Marianne [DK/DK];

Novo Nordisk a/s. Novo AUc, DK-2880 Bagsvterd (DK).
VIND, Jesper [DK/DK]; Novo Nordisk a/s. Novo Alle\
DK-2880 Bagsvand (DK). HALKIER, Torben [DK/DK];
Novo Nordisk a/s, Novo A116, DK-2880 Bagsvaerd (DK).
J0RGENSEN, Steen, Troels [DK/DK]; Novo Nordisk a/s.
Novo AIM, DK-2880 Bagsvaerd (DK).

(74) Common Representative: NOVO NORDISK A/S; Corporate
Patents, Novo Alle\ DK-2880 Bagsvaerd (DK).

(81) Designated States: AL, AM, AT, AU, AZ, BB, BG, BR, BY,
CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL,
IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV,
MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO t RU,
SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG f US, UZ,
VN, ARIPO patent (KE, LS, MW, SD, SZ, UG), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT,
LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI,
CM, GA, GN, ML, MR, NE, SN, TD, TG).

Published

With international search report

(54) Title: A MODIFIED ENZYME WITH LIPOLYTIC ACTIVITY
(57) Abstract

The invention relates to a modified enzyme with lipolytic activity recovered from a filamentous fungi or a bacteria having one or
more peptide additions at the N-terminal and/or the C-teiminal ends in comparison to the parent enzyme. Further, the invention relates to
a DNA sequence encoding said modified enzyme, a vector comprising said DNA sequence, a host cell harbouring said DNA sequence or
said vector, and a process for producing said modified enzyme with lipolytic activity.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under the PCT.

Armenia

United Kingdom

Malawi

Austria

Georgia

Mexico

Australia

Guinea

Niger

Barbados

Belgium

Hungary

Norway

Burkina Ftso

Ireland

New Zealand

Bulgaria

Italy

Poland

Bcnjo

Japan

Portugal

Bnzil

Kenya

Romania

Behras

Kyrgystan

Russian Federation

Canada

Democratic People's Repubbc

Sudan

Central African Republic

of Korea

Sweden

Congo

RepnbGc of Korea

Singapore

Switzerland

Slovenia

Cfte<n voire

Slovakia

Cameroon

Sri Lanka

China

Liberia

Swaziland

CzccfaoskTvaJua

Chad

Czecb Republic

Togo

Genmfjr

Lama

Tajikistan

Denmark

Monaco

Trinidad and Tobago

Estonia

Repcbtic of Moldova

Ukraine

Span

Madagascar

Uganda

Rnbnd

Mali

United States of America

France

Mongolia

Uzbekistan

Gabon

Mauritania

WO 97/04079

PCT/DK96/00322

Title: A modified enzyme with lipolytic activity
FIELD OF THE INVENTION

The present invention relates to a modified enzyme with lipolytic activity, a DNA
sequence encoding said modified enzyme, a vector comprising said DNA sequence, a host cell
5 harbouring said DNA sequence or said vector, and a process for producing said modified
enzyme with lipolytic activity.

Further the invention relates to a method for applying a peptide addition to a parent
enzyme with lipolytic activity, a composition comprising the modified enzyme with lipolytic
activity of the invention, the advantageous use of the modified enzyme of the invention in
10 detergent compositions, and further a method for improving the washing performance of
detergent compositions.

BACKGROUND OF THE INVENTION

Detergent enzymes have been marketed for more than 20 years and are today well
is established as normal detergent ingredients in both powder and liquid detergent all over the
world.

Detergent compositions may comprise many different enzymes, of which proteases,
amylases, cdlulases, lipases, cutinases are the most important today.
Lipolytic enzymes

20 lipolytic enzymes (i.e. enzymes classified under the Enzyme Classification number E.C.
3.1.1 (Carboxylic Ester Hydrolases) in accordance with the Recommendations (1992) of the
International Union of Biochemistry and Molecular Biology (IUBMB)) are enzymes which
can be used for removing lipid or fatty stains from clothes and other textiles.

Various microbial lipases have been suggested as detergent enzymes. Examples of such

25 lipases include a Humicola lanuginosa lipase, e.g. described in EP 258 068 and EP 305 216,
a Rhizomucor miehei lipase, e.g. as described in EP 238 023, Absidia sp. lipolytic enzymes
(WO 96/13578), a Candida lipase, such as a C. amaraica lipase, e.g. the C. antaraica lipase
A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P.
pseudoalcaligenes lipase, e.g. as described in EP 218 272, a P. cepacia lipase, e.g. as

30 described in EP 331 376, a Pseudomonas sp. lipase as disclosed in W095/ 14783, a Bacillus
lipase, e.g. a B. subtilis lipase (Dartois et al., (1993) Biochemica et Biophysica acta 1131,
253-260), a B. stearothermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO
91/16422).

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

Furthermore, a number of cloned lipases have been described, including the PeniciUium
camembertii lipase described by Yamaguchi et al., (1991), Gene 103, 61-67), the Geotricwn
amdidwn lipase (Schimada, Y. et al., (1989), J. Biochem., 106, 383-388), and various
Rhizopus lipases such as a/?, delemar lipase (Hass, MJ et al., (1991), Gene 109, 117-113) a
5 R. nxveus lipase (Kugimiya et al., (1992), BioscL Biotech. Biochem. 56, 716-719) and a R.
oryzae lipase.

Other types of lipolytic enzymes having been suggested as detergent enzymes include
cutinases, e.g. derived from Pseudomonas mendocina as described in WO 88/09367, or a
cutinase derived from Fusarium sokmipisi (e.g. described in WO 90/09446).
10 In recent years attempts have been made to prepare modified lipolytic enzymes, such as
variants and mutants having improved properties for detergent purposes.

In most cases lipolytic enzymes with improved washing performance have been
constructed by site-directed mutagenesis resulting in substitution of specific amino acid
residues which have been chosen either on the basis of their type or on the basis of their
15 location in the secondary or tertiary structure of the mature enzyme.

An alterative general approach for modifying proteins and enzymes have been based on
random mutagenesis, for instance, as disclosed in US 4,894,331, WO 93/01285, and WO
95/22615.

20 Comments to prior art

It is known from prior art to modify lipolytic enzymes by site-directed mutagenesis to
obtain an improved performance, inparticular washing performance of lipolytic enzymes. The
generally used concept has been to insert, delete or substitute amino acids within the structural
part of the amino acid chain of the parent lipolytic enzyme in question. Lipolytic enzymes
25 with a significantly improved washing performance have been achieved this way.

However, there is a need for providing lipolytic enzymes with an even further improved
performance, such as washing performance and/or even further improved dishwashing
properties than the lipolytic enzymes prepared by these prior art methods.

30 SUMMARY OF THE INVENTION

Thus, the object of the present invention is to improve properties of enzymes with
lipolytic activity, in particular to improve the washing performance of such enzymes.

It has surprisingly been found that it is possible to significantly enhance the washing

SUBSTITUTE SHEET (RULE 26)

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performance of a lipolytic enzyme by applying a peptide addition to the N- and/or C-terminal
of the enzyme.

Consequently, in a first aspect the invention relates to a modified enzyme with lipolytic
activity which as compared to its parent enzyme has one or more peptide additions in its C-

5 terminal and/or N-terminal end.

In the present context the term "peptide addition" is intended to indicate that a stretch of
one or more consecutive amino acid residues has been added to either or both of the N- and/or
C-terminal end(s) of the parent enzyme or inserted within the non-structural part of the N-
and/or C-terminal end(s) of the parent enzyme.

io The term "non-strucural part" is intended to indicate the part of the N- and C-terminal
end, respectively, which is outside the first or last, respectively, structural element, such as an
ct-helix or p-sheet structure, of the folded mature enzyme. The non-structural part may easily
be identified in a three-dimensional structure or model of the enzyme in question. Typically,
the non-structural part comprises the first or the last about 1-20 amino acid residues of the

is amino acid sequence constituting the enzyme.

The term "mature enzyme" is used in its conventional meaning, i.e. to indicate the active
form of the enzyme resulting after expression and posttranslational processing (to remove pro
and/or pre-sequences) by the producer organism in question. When the enzyme is a secreted
enzyme, the mature enzyme will normally be the form of the enzyme resulting after secretion.

20 More specifically this means that the pre- and pro-peptide sequences, if present, have been
removed from the initially translated enzyme, i.e. the unprocessed enzyme.

The term "parent* enzyme in intended to indicate the enzyme to be modified according to
the invention. The parent enzyme may be a naturaliy-occuring (or wild type) enzyme or may
be a variant thereof prepared by any suitable means. For instance, the parent enzyme may be

25 a variant of a naturally-occurring enzyme which has been modified by substitution, deletion or
truncation of one or more amino acid residues or by addition or insertion of one or more
amino acid residues to the amino acid sequence of a naturally-occurring enzyme, typically in
the structural part of the enzyme.

In other aspects the invention relates to a DNA sequence encoding a modified lipolytic

30 enzyme as defined above, a recombinant vector or transformation vehicle comprising a DNA
sequence of the invention, a host cell harbouring a DNA sequence of the invention or a vector
of the invention, and a process for preparing a modified lipolytic enzyme by cultivation of
said host cell.

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

The modified lipolytic enzyme of the invention may conveniently be used as a detergent
enzyme and accordingly, in final aspects the invention relates to a detergent additive or a
detergent composition comprising a modified lipolytic enzyme of the invention.

5 BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows the nucleotide and amino acid sequence of the coding region of the Hwmcola
lanuginosa lipase gene as present in the yeast expression vector pIS037. The signal sequence
(amino acids 1 to 17) is the original signal sequence from Hwmcola lanuginosa. The SPIRR
peptide addition is located at amino acid residue 18 to 22. Amino acid residue 23 (E) is the

10 first amino acid residue of the parent lipase expressed in Aspergillus oryzae.

Figure 2 shows the nucleotide and amino acid sequence of the coding region of the Hwmcola
lanuginosa lipase gene as present in the £. coli expression vector pJSQ215. The signal
sequence (amino acids 1 to 20) is the A tyricus protease I signal (WO 96/17943). The SPIRR
peptide is added after amino acid residue 20. Amino acid residue 26 (E) is the first amino arid

15 residue of the parent lipase expressed in Aspergillus oryzae.

Figure 3 shows the nucleotide and amino arid sequence of the coding region of the Hwmcola
lanuginosa lipase gene as present in the E. coli expression vector pSX58L The signal
sequence (amino acids 1 to 20) is the A. tyricus protease I signal sequence (WO 96/17943).
Amino acid residue 21 (E) is the first amino acid residue of the parent lipase.

20 Figure 4 the construction of pSX164;
Figure 5 the construction of pSX578;
Figure 6 the construction of pSX581;
Figure 7 shows the plasmid pSX581;
Figure 8 shows the plasmid pJS037;

25 Figure 9 shows the construction of Aspergillus vector pCaHj485

DETAILED DESCRIPTION OF THE INVENTION
Peptide addition

30 As stated above it has surprisingly been found that a significantly improved wash
performance of lipolytic enzymes may be achieved when an appropriate peptide addition is
applied to a non-structural part of the enzyme in its mature form or at the C-terminal and/or
N-terminal end of the mature enzyme.

SUBSmUTE SHEET (RULE 2Q

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PCT/DK96/00322

The term "improved wash performance" is intended to indicate that the modified enzyme
of the invention has a better lipid soil removing capability than the unmodified parent enzyme
when tested under wash like conditions. The improvement is often indicated in terms of u an
improvement factor" (f^, (further reference vide the Materials and Methods section
further below). Dependent on the peptide addition and the mature enzyme an improvement
fector (fimp^c) ) in the range of 1-5 , or even up to 10 (such as in the range of 1-10) has been
obtained. It is presently believed that even higher improvement factors such as up to 20, even
up 30, or even up to 50, such as between 30 and 50, or even higher may be achieved in
accordance with the present invention.

The term u an appropriate peptide addition" is used to indicate that the peptide addition to
be used is one which is capable of effecting an improved wash performance. The
"appropriateness" of the peptide addition may be checked by a comparative analysis of the
wash performance of a modified enzyme to which the peptide addition has been applied and
of the corresponding parent enzyme, respectively. The wash performance may, e.g., be
determined by any suitable technique such as any of the wash performance assays described in
the present application.

It is presently contemplated that the improved wash performance effected by the peptide
addition is, at least in part, due to an increased affinity of the modified lipolytic enzyme
towards its lipid substrate (although this may not be the only reason).

The present invention is not limited to improving the wash performance of a parent
lipolytic enzyme. It is contemplated that also other properties of parent lipolytic enzymes may
be improved in accordance with the present invention, i.e. by applying an appropriate peptide
addition at or within a non-structural part of the C-terminal and/or N-terminal end of the
parent enzyme. More specifically, it is contemplated that the activity of a parent lipolytic
enzyme, e.g., in removing pitch in the paper and pulp industry, in degreasing hides in the
leather industry, in acting as a catalyst in organic syntheses, etc., may be significantly
improved by applying an appropriate peptide addition at or within the N-terminal or C-
terminal end of a lipolytic enzyme, i.e. a peptide addition which is capable of exerting the
desired function. Also in these connections it is believed that the improved activity may be at
least partly due to an improved affinity for the substrate in question.

As a consequence of the improved activity it may be possible to reduce the dosage of the

enzyme required for a given purpose considerably, as compared to the dosage of needed

dosage of the unmodified parent enzyme.

RECTIFIED SHEET (RULE 91)

WO 97/04079

PCI7DK96/00322

In the context of the present invention the term "modified enzyme" is intended to indicate
a derivative or a variant of a parent enzyme, which derivative or variant as compared to the
parent enzyme comprises a peptide addition at the C-terminal and/or N-texminal end (fused to
the first and/or last amino acid residue of the parent enzyme) and/or within the non-structural

5 part of the C- and/or N-teminal end of the parent enzyme. Thus, for instance, the modified
enzyme may comprise a peptide addition at either the N-terminal or the C-terminal end or
both in the N- and the C-terminal ends of the parent lipolytic enzyme.

It is presently believed that the capability of the peptide addition of providing the desired
effect (such as improved wash performance, improved performance in degreasing of hides,

to etc, depends on, e.g., the identity of the parent enzyme to be modified, the structure
(including length) of the peptide addition, the impact of the peptide addition on the structure
of the entire lipolytic enzyme, the nature or functionality of amino acid residues of the peptide
addition, etc. A prerequisite for the peptide addition being capable of providing the desired
effect is, of course, that the modified enzyme containing the peptide addition is expressible in

is a suitable host organism. The following general considerations may be of relevance for the
design of a suitable peptide addition:

Length of peptide addition: It has been found that peptide additions containing varying
numbers of amino add residues are capable of providing the desired effect and thus, it is not
possible to specify an exact number of amino acid residues to be present in the peptide

20 addition to be used in accordance with the present invention. It is contemplated that the upper
limit of the number of amino acid residues is determined, inter alia, on the impact of the
peptide addition on the expression, the structure and/or the activity of the resulting modified
enzyme. It is believed that the peptide addition may comprise a substantial number of amino
acid residues, however, without all of these amino acid residues need to contributing to the

25 desired effect (even if the peptide addition contains a substantial number of amino acid
residues only a small number of these need to providing the desired function, this small
number may be termed the functional part of the peptide addition). The main consideration in
relation to the lower limit of the number of amino acid residues of the peptide addition will
normally be that the number should be sufficient to provide the desired effect

30 The peptide addition may thus comprise a single amino acid residue or an amino add
chain of from 2 and 500 amino adds, such as from 1 to 200, or from 2 to 100, preferably
from 2 to 50, such as 3 to 50, even more preferably from 7-45 and still more preferably
between 1 and 15, such as between 1 and 10 or 1 and 7, especially between 4 and 10, such as

SUBSTITUTE SHEET (RULE 26)

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PCT/DK96/00322

4 and 7 amino acids.

Stability: The peptide addition should preferably be chosen so as to provide a modified
lipolytic enzyme with an acceptable stability (e.g. structural stability and/or expression
stability) or so as to not significantly reduce the structural stability of the parent enzyme.

5 Although many peptide additions are not believed to confer any substantial structural
instability to the resulting modified enzyme, it may in certain instances and with certain parent
enzymes be relevant to choose a peptide addition which in itself can confer a structural
stability to the modified lipolytic enzyme. For instance, a peptide addition which in itself
forms a structural element, such as an a-helix or a (5-sheet, may stabilize the resulting

to modified enzyme and thus be used in the context of the present invention. Peptide sequences
capable of forming such structures arc known in the art. Alternatively, an improved structural
stability may be provided by introduction of cy stein bridges in the modified lipolytic enzyme
of the invention. For instance, a cystein bridge between the peptide addition and the mature
part of the enzyme may be established if at least one of the amino acid residues of the peptide

is addition is a cystein residue which is located so as to be able to form a covalent binding to a
cystein residue in the mature part of the enzyme. The positive effect of introducing a cystein
bridge is illustrated in Example 19. If no suitable cystein is present in the mature enzyme, a
cystein may be inserted at a suitable location of said parent enzyme, conveniently by replacing
an amino acid of the parent enzyme, which is considered unimportant for the activity. *

20 In addition, it may be desirable that at least one of die amino acid residues of the peptide
addition is chosen so as to make the peptide addition less susceptibility to proteolytic
degradation by proteolytic enzymes of the host cell used for expressing the modified lipolytic
enzyme. For instance, the peptide addition may comprise at least one, and preferably at least
two proline residues. Preferably, the peptide addition comprises 1-5, such as 1-4 or 1-3 or

25 two or one proline residues. The proline residue(s) is(are) preferably placed at the proteolytic
cleavage site or close thereto. Alternatively, the peptide addition may be one which provides a
protease stable loop to the modified lipase, e.g. as described in EP 407 225 or WO 93/1 1254.

Nature of amino acid residues of the peptide addition: As stated above and without being
limited to any theory, it is presently believed that the improved performance may at least

30 partly be due to an increased affinity of the modified lipolytic enzyme toward the substrate
provided by the peptide addition. In particular in relation to wash performance, it is believed
that favourable electrostatic interactions may be obtained between the negatively charged lipid
surface and positively charged and/or hydrophobic amino acid residues present in the

SUBSTITUTE SHEH

WO 97/04079

PCI7DK96/00322

modified enzyme. Accordingly, it is particularly preferred that the modified enzyme of the
invention comprises a peptide addition with at least one positive charge, such as at least 2, 3,
4 or more positive charges or expressed differently, in which a substantial number of the
amino acid residues of the peptide addition is positively charged and/or hydrophobic.
5 Analogously, and in order to reduce the negative charge in a non-structural end of the
parent enzyme it is preferred to remove at least one such as two or more negatively charged
amino acid residues from a non-structural N-terminal or C-terminal part of the parent enzyme
of choice, in particular from the part of the parent lipase being constructed of the 1-5 first or
last N-terminal or C-terminal amino acid residues, such as 1-4, or 1-3 or 1-2. The negatively

10 charged amino acid residue may either be removed or replaced by a neutral, a positively
charged or a hydrophobic amino add residue. For instance, the negatively charged amino acid
residue to be removed may be an E or D which may be replaced with either of the positively
^ charged amino acid residues R, K or H, the neutral amino acid residues S, T, G or Q, or die
hydrophobic amino acid residues A, I, W F or L. Similarly, a neutral amino acid residue of a

is non-structural N-terminal or C-terminal part of the parent enzyme may be replaced with a
positively charged or hydrophobic amino acid residue as defined above.

Accordingly, the modified lipolytic enzyme of the invention in addition or as an
alternative to a N-terminal and/or C-terminal extension may comprise a mutation in the non-
structural C-terminal and/or N-terminal end of the parent enzyme, which mutation has

20 involved deleting or replacing a negatively charged amino acid residue of said non-structural
part with a positively charged or neutral amino acid residue or with a hydrophobic amino acid
residue.

If a peptide addition is present in both the N- and the C-terminal of the parent enzyme,
the peptide addition at or within each of the terminals may have the same or a different amino
25 arid sequence.

Test of suitability of peptide addition: the effect of using a given peptide addition, e.g.,
designed on the basis of the above principles may be tested by constructing a modified
lipolytic enzyme containing the peptide addition and testing the properties of the resulting
enzyme for die desired enzyme application such as wash, pitch removal, degreasing of
30 leather, etc either in a full scale test or in an assay which correlates well with the enzyme
application in question.

The peptide addition can be generalised in the following way.

The first residue (counted from the outer residue) is named tt a w , the second is named "b",

SUBSTITUTE SHEET

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PCT/DK96/00322

the third tt c" etc. Thus, in case of an N-terminal addition the first amino acid residue is

termed V, in case of a C-terminal addition the last amino acid residue is termed tt a B .

In an important embodiment of the invention the peptide addition consists of from 1 to 7

amino adds. Such peptide addition, which can be applied to both the N- and/or C-terminal of
5 the parent enzyme, can be referred to as:

a (one amino acid peptide addition)

a-b (two amino acids peptide addition)

a-b-c (three amino adds peptide addition)

a-b-c-d (four amino acids peptide addition)
10 a-b-c-d-e (five amino acids peptide addition)

a-b-c-d-e-f (six amino acids peptide addition)

a-b-c-d-e-f-g (seven amino acids peptide addition)

Each letter defines an amino acid residue.

a, b, c, d, e, f and g may independently be any amino acid including Alanine (A), Valine (V),
Leucine (L), Isoleucine (I), Proline (P), Phenylalanine (F), Tryptophan (W), Methionine (M),
Glycine (G), Serine (S), Threonine (T), Cysteine (Q, Tyrosine (Y), Asparagine (N),
Glutamine (Q), Aspartic arid (D), Glutamic arid (E), Lysine (K), Arginine (R), and Hfetidine
20 (H).

In specific embodiments a, b, c, d, e, f, and g are independently one of the following
amino acids:

a: Leu, Be, Val, Trp, Phe, Ser, Arg, Cys, or Lys,

b: Leu, He, Val, Tip, Phe Ser, Pro, Arg, Lys, Cys or His,
25 c: Leu, lie, Val, Trp, Phe, Ser, Pro, Arg, Cys, or Lys.

d: Leu, He, Val, Tip, Phe Ser, Pro, Aig, Cys, or Lys.

e: Leu, He, Val, Trp, Phe, Pro, Arg, Lys, Ala, Glu, Cys, or Asp,

f: Leu, De, Val, Tip, Phe, Pro, Arg, Lys, Ala, Glu, Cys, or Asp,

g: Leu, De, Val, Tip, Hie, Pro, Arg, Lys, Cys, or Met
30 In a preferred embodiment at least one such as (me, two, three or four of a, b, c, d, e, f,

or g is a positively charged amino arid, Le. Arg (R) or Lys (K) or a hydrophobic amino acid,

Le. Lai, He, Val, Tip or Phe.

As stated further above, and dependent cm the host cell of choice it is generally believed

SIBSTITUTE SHEET (RULE 28)

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PCI7DK96/00322

that it is important that the peptide addition comprises at least one proline residue in order to
protect the modified lipolytic enzyme against proteolytic degradation during the processing of
the enzyme by the host cell of choice. It may be desirable that die proline residue occupies
position two (i.e. b) and/or three (i.e. c) of the peptide addition or a position close to die
5 desired cleavage point (i.e. the point where processing by the host cell in question is believed
to occur). Accordingly, in one embodiment b and optionally c of die peptide addition is Pro

In another embodiment of the invention a-b is SP (Ser-Pro), A-P or Q-P. If the peptide
addition contains more amino acid residues, e.g. between 4 and 7 amino acids the peptide
addition has the general formula SPcd, SPcde, SPcdef, SPcdefg or APcd, APcde, APcdef,
10 Apcdefg or QPcd, QPcde, QPcdef, QPcdefg. In each of these formulae c, d, e , f, and g may
be any amino arid. However, preferred are the above mentioned group of amino adds.

In another embodiment a-b comprise at least one positive amino adds (i.e. Arg and Lys)
or hydrophobic amino arid residue (Le. Leu, De, Val, Trp and Phe).

Specifically, the peptide addition applied to the parent lipolytic enzyme may
is advantageously be one of the following amino arid residues or peptides:
Arg (R), or Lys (K), or Leu (L), or De (I), or
Val (V), or Trp (W) or Phe (F), or
Arg-Pro (RP), or
Lys-Lys (KK), or
20 Arg-Lys (RK), or
Lys-Arg (KR), or
Arg-Arg (RR), or
Arg-Arg-Pro (RRP), or
Arg-Pro-Val-Ser-Gln-A^) (RPVSQD)
25 Ser-Pro-De-Arg-Met (SPIRM), or
Ser-ProDe-Arg-Ala-Arg (SPIRAR), or
Ser-Pro-Ile-Arg-Pn>-Arg (SPIRPR) or
Ser-Pro-De-Arg-Glu-Arg (SPIRER), or
Ser-Pro-Ile-Arg-Lys (SPIRK), or
30 Ser-Pro-Ile-Lys-Lys (SPIKK), or
Ser-Pro-Ile- Arg-Arg-Pro (SPIRRP), or
Ser-Pro-Pro- Arg-Arg (SPPRR), or
Ser-Pro-Iso-Pro-Arg (SPIPR), or

SUBSTTFUTE SHEET (RULE 26)

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Ser-Pro-Arg-Pro-Arg (SPRPR), or

Ser-Pro-Ee-Arg (SPIR), or

Ser-Pro-De-Arg-Arg (SPffiR), or

Ser-Cys-ile-Arg-Arg, (SC3RR), or
5 Ser-Pro-ne-Arg-Pro-Arg-Pro (SPKPRP), or

Ser-Cys-Ee-Arg-Pro-Arg-Pro (SCPIRPRP), or

Ser-Pro-Arg-Arg-Pro-Arg-Thr (SPRRPRT), or

Ser-Pro-Phe-Arg-Pro-Lys-Leu (SPFRPKL), or

Ser-Pro-Pro-Arg-Arg-Pro (SPPRRP), or
10 Ser-Pro-De-Arg-Arg-Glu (SPIRRE), or

Ser-Pro-Pro-Arg-Pro-Pro (SPPRPP), or

Ser-Pro-Pro-Arg-Pro-Arg (SPPRPR), or

Ser-Pro-Pro-Trp-Trp-Pro (SPPWWP), or

Ser-Pro-Pro-Trp-Arg-Pro (SPPWRP), or
15 Ser-Pro-Pro-Arg-Trp-Pro (SPPRWP), or

Ser-Pro-Pro-Arg-Trp-Pro (SPPRWP), or

Ser-His-Trp-Arg-Arg-Trp (SHWRRW), or

Ser-His-Tip-Arg-Lys (SHWRK), or

Ser-His-Trp-Arg-Arg (SHWRR), or
20 Thr-Ala -Le-Arg-Pro-Arg-Lys (TAIRPRK),

Ser-Thr-Arg-Arg-Pro-Arg-Pro (STRRPRP),

Gly-Pro-De-Arg-Pro-Arg-Pro (GPIRPRP), or

Leu-Pro-Phe-Arg-Glu-Arg-Pro (LPFRQRP), or

Ser-Arg-Ser-Arg-His-Asp-Ala (SRSRHNA), or
25 ne-Pro-De-Arg-Pro-Arg-Arg (BPIRPRR), or

Ser-Thr-Arg-Arg-Pro-Arg-Pro (STRRPRP), or

Thr-Ala-Ile-Arg-Pro-Arg-Lys (TAIRPRK), or

Trp-Arg-Trp-Arg-Trp-Arg (WRWRWR), or

Gh>Pro-Ile-Arg-Arg (QPIRR), or
30 Ser-His-Trp-Glu-Glu (SHWQQ), or

Ser- Ala-Leu-Arg-Pro-Arg-Lys (SALRPRK).

Also contemplated according to the invention is additions comprising more than 7 amino

acids, such as from 8 to IS amino acids.

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Such peptides can be generalised as:
a-b-c-d-e-f-g-h (8 amino acid peptide)
a-b-e-d-e-f-g-h-i (9 amino acid peptide)
a4>-c-d-e-f-g-lw-j (10 amino acid peptide)
5 a-b-c-d-e-f-g-h-i-j-k (1 1 amino acid peptide)
a-b-od-e-f-g-h-i-j-k-l (12 amino acid peptide)
a-b-c-d-e-f-g-h-i-j-k-l-m-(13 amino acid peptide)
a-b-c-d-e-f-g-h-i-j-k+m-n(14 amino arid peptide)
a-b-c-d-e-f-g-h-i-j-k-l*m*n-o (15 amino acid peptide).

a to o may be any of the twenty amino acids mentioned above.

The a-g stretch may be as defined above in relation to a peptide addition comprising 1 to
7 amino acid residues.

h, i, j, k, 1, m, n, o may as mentioned above be any amino arid, preferably any of the
15 following amino acids: Arg, Lys, Ala, Val, Trp, lie, Phe, Ser or Pro.
Specific examples of such additions are listed below:

Arg-Pro-Arg-Pro-Arg-Pro-Arg-Pro (RPRPRPRP), or

Ser-Ser-Thr-Arg-Aig-Ala-Ser-Pn>Ile-Lys-Lys (SSTRRASPKK), or

Ala-Trp-Txp^Pro-Ser-Pro-De-Arg-Pro-Arg-Pro (AWWPSPIRPRP), or
20 Ala-Pro-Pro-Pro-Arg-Pn>Arg-Pro-^ (APPPRPRPRPRP), or

Ak-Pro-Pro-Pro-Arg-Thr-Arg-I^Arg-P^Arg-Ser (APPPRTRPRPRS), or

Ser-Pro-Lys-Arg-Lys-Pro-Arg-Pro (SPKRKPRP), or

Ser-Gln-Arg-ne-Lys-Gln-Arg-Ile-Lys (SQRIKQRIK), or

Ser-Pro-Pro-Pro-Arg-Pro-Arg-Pro (SPPPRPRP), or
25 Ser-Pro-De-Arg-Pro-Arg-Pro-Arg-Pro-Arg SPIRPRPRPR, or

Ser-Pro-Ee-Arg-Lys-AlarTip-Trp-Pro (SPIRKAWWP), or

Ala>Pro-Pro-Pro-Lys-Ala-Ser-Pro-Aig<3b-Arg-Pro (APPPKASPRQRP), or

Ser-Pro-De-Arg-Pro-Arg-Pro-Se^^ (SPIRPRPSPIRPRP), or

Ser-Pro-Pro-Aig-Trp-Pro-Arg-Arg (SPPRWPRR), or
30 Ser-Pro-Pro-Arg-Tip-Pro-Aig-Tip (SPPRWPRW), or

Ser-Pro-Pno-Arg-Tip-Pro-Trp-Arg (SPPRWPWR), or

SCT-Pro-Pro-Trp-Arg-Pro-Arg-Arg (SPPWRPRR), or

SCT-Pro-Pro-Trp-Trp-Pro-Arg-Trp (SPPWWPRW, or

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Ser-Pro-Pro-Trp-Trp-Pro-Trp-Arg (SPPWWPWR), or
Ser-Pro-Pro-Trp-Trp-Pro-Trp-Trp (SPPWWPWW), or
Ser-Pro-Pro-Tip-Pro-Arg-pro-Arg-Pio (SPPWPRPRP), or
Ala-I^Pro-Pro-Arg-Pro«Arg-Leu-Leu-Pro-Ile-SCT (APPPRPRLLPIS), or

5 Ala-Pro-Pn>Pro-Thr-Arg<Jln-Ai^<5b-Ser-Pro (APPPTRQRQSP), or
Ala-Pro-Pm*Pro-Arg-Thr-Ee-Pix>-Arg-Ser-Ser-Pro (APPPRHPRSSP).

In any of the above specified peptide additions (whether comprising 1 to 7 or 1 to 15
amino arid residues) in which the position "a" is a Ser, Ala, Arg, Lys or Pro, the Ser may be
replaced with an Ala, Arg, Lys or Pro, the Ala with a Ser, Arg, Lys or Pro and the Arg, Lys

10 or Pro with a Ala or Ser.

It is to be emphasised that the above peptide addition may be at either the N-terminal
and/or the C-terminal. Examples of modified lipolytic enzymes with both a N- and a C-
terminal peptide addition include all combinations of the peptide additions specifically
mentioned above. Two specific examples of such are the N-terminal addition SPERPRP

is together with the C-terminal addition RRP or RR.

If the peptide addition is inserted into the non-structural part of the parent enzyme, it may
replace one or more of the amino acid residues of said non-structural part. For instance, the
peptide addition may replace one or more amino arid residues occupying the first, e.g. 1-5,
amino acid residues of the N-terminal end and/or the last, e.g. 1-5, amino acids t>f the

20 enzyme (i.e. the 1-5 amino acid residues of the C-terminal end). For instance, the peptide
addition may replace amino arid residue(s) 1 and/or 2 and/or 3 and/or 4, and/or 5, etc. from
either end of the parent enzyme.

When the parent enzyme is H. lanuginosa lipase it has been of particular interest to combine
any of the above peptide additions (applied in the N-terminal) with a deletion of the parent
25 first (IE).

In accordance with the invention, it is also contemplated to apply, to the modified
enzyme, one or more charged amino acids which permit effective purification of the modified
enzyme. Techniques for doing this is well known by a person skilled in the art of molecular
biology.

Methods of applying a peptide addition to a parent lipolytic enzyme

Although a modified enzyme of the invention may be obtained by adding (fusing or
inserting) a synthetically produced peptide addition into the parent lipolytic enzyme in

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question, it is presently preferred that the modified enzyme of the invention is prepared by i)
modifying the nucleotide, preferably DNA, sequence encoding the parent enzyme so as to
encode the desired peptide addition applied to the N- and/or the C-terminal end(s) of the
parent enzyme (e.g. by inserting a nucleic arid (preferably DNA) sequence encoding the
5 peptide addition at the relevant location in the nucleic acid (preferably DNA) sequence
encoding the parent enzyme), ii) expressing the resulting modified nucleic acid (preferably
DNA) sequence in a suitable expression system, and iii) recovering the resulting modified
enzyme.

In the present context, the term "applied to" is intended to indicate that the addition is
10 fused to the N- and/or C-terminal end (e.g. to the first or last amino arid residue) of the
mature enzyme or inserted into a non-structural part of the N-terminal and/or C-terminal end
of the mature enzyme.

Many enzymes are expressed as "prepro-enzymes", i.e. as enzymes consisting of the
mature enzyme, a secretory signal peptide (i.e. prepeptide) and a pro-peptide. The prepro-

15 enzyme is processed intracellulariy to be secreted into the fermentation medium, from which
the mature enzyme can be isolated and/or purified. The peptide addition to the parent enzyme
can be carried out by applying nucleic arid sequences encoding the desirable peptide additions
upstream (for N-terminal peptide additions) and/or downstream (for C-terminal peptide
additions) to the DNA sequence encoding the parent enzyme.

20 The insertion should be performed in such a way that the desired modified enzyme (i.e.
having the desired peptide addition(s)) is expressed and secreted by the host cell after
transcription, translation, and processing of die enzyme. The term "processing" means in this
context removal of pre- and pro-peptides (except, of course, when the pro-peptide is identical
to the desired peptide addition. This will be dealt with furtehr below).

25 Downstream sequences (encoding a C-terminal addition) can be inserted between the
DNA sequence encoding the parent enzyme and the terminating codon. However, if the
unprocessed DNA sequence comprises a pro-peptide encoding DNA sequence at the C-
terminal end die insertion/addition of die DNA sequence encoding the peptide addition can
also take place between the DNA sequences encoding the pro-peptide and the mature enzyme,

30 respectively.

In most cases it is possible to extend die parent enzyme upstream by inserting a DNA
sequence encoding the peptide addition between the DNA sequence encoding the pro-peptide
or the prepeptide (if no prosequence is present) and the DNA sequence encoding die mature

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enzyme.

The insertion/addition of a DNA sequence encoding the peptide addition can be carried
out by any standard techniques known by any skilled person in the field of molecular biology,
cf., e.g. Sambrook et al., 1989). This include, e.g., the polymerase chain reaction (PCR)

5 using specific primers, for instance described in US patent 4,683,202 or R.K. Saiki et aL,
(1988), Science, 239, 487-491. How to provide for the expression and secretion of adjacent
DNA sequenced) will be described below.

In connection with the present invention it has been found that some host cells may be less
suited for the production of a desired modified enzyme, in that part or all of the peptide

10 addition(s) may be cut off during the posttranslational or other processesing performed by the
host cell. Accordingly, the term "suitable expression system" is intended to indicate an
expression system (host cell and optionally expression vector) which allows for at least a
portion of an intact desired modified enzyme to be produced, i.e. an expression system which
does not, e.g. as part of the posttranslational or other processing by the host cell of choice,

15 remove part or all of the peptide addition (and thereby produce the enzyme without the
desired peptide addition). Typically, the expression system to be used is devoid of one or
more proteolytic activities exerting the undesired posttranslational processing. The choice of
expression system and thus host cell will depend on the lipolytic enzyme to be produced as
will be discussed in detail further below.

20 While care must be exerted to select a proper expression system for producing a modified
enzyme of the invention (in particular when a modified DNA sequence is used for the
production), it has been found that a modified lipolytic enzyme according to the invention
(having an improved wash performance) may be obtained by expressing a DNA sequence
encoding the parent lipolytic enzyme in question in an expression system which is incapable of

25 processing the translated polypeptide in the normal manner, and thereby results in the
production of an enzyme which comprises a part of or the entire propeptide or a similar
peptide sequence associated with the mature protein prior to its processing. In this case, the
propeptide or similar peptide sequence constitutes the peptide addition. The propeptide or
similar peptide sequence may be heterologous or homologous to the parent enzyme and can be

30 present in both the N- and C-terminal of the parent enzyme. The production of a modified
lipolytic enzyme according to the invention using this latter technique is described further
below.

Accordingly, if a suitable stretch of amino acids is already encoded in the prepro form of

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the parent enzyme and this stretch of amino adds is cut off in the processing of the enzyme by
a given expression system, the peptide addition can be applied by changing the expression
host system to a system in which said processing of said stretch of amino acids does not
occur. In such a case the secretory signal pre-peptide will be cut off during or after the
secretion, resulting in a modified enzyme consisting of the parent enzyme comprising the pro-
peptide or part thereof or a similar peptide sequence encoded by the corresponding DNA
sequence, i.e. a lipolytic enzyme being extended at either its N-terminal or C-terminal end.

In other words, in a further aspect the invention relates to a method for increasing the
wash performance or other activity of a parent enzyme, comprising the steps of:

a) introducing a DNA sequence encoding the parent lipolytic enzyme into an expression
vector,

b) introducing said DNA sequence or expression vector into a host cell incapable or
inefficientl in the processing of the expressed pro-enzyme to the mature enzyme,

c) cultivating said host cell under conditions suitable for production of the enzyme comprising
a part of the entire pro(pre)-sequence, and

d) recovering and optionally purifying the resulting modified enzyme.

Yeast cells have been found of particular use for applying peptide additions On the form of the
propeptide or a part thereof) to parent fungal lipolytic enzymes, in particular the H.
lanuginosa lipase enzyme.

In an alternative and highly preferred embodiment the peptide addition is designed and
applied by means of random mutagenesis according to the following principle:

a) subjecting a DNA sequence encoding the parent lipolytic enzyme with a pqtfide addition to
localized random mutagenesis in the peptide addition or in a non-structural part of the C-
terminal or N-terminal end of the parent enzyme,

b) expressing the mutated DNA sequence obtained in step a) in a host cell, and

c) screening for host cells expressing a mutated lipolytic enzyme which has an improved
performance as compared to the parent lipolytic enzyme.

By this approach a number of highly advantageous peptide additions have been created.
The localized random mutagenesis may be performed essentially as described in WO
95/22615. More specifically, the mutagenesis is performed undo- conditions in which only
one or more of the above areas are subjected to mutagenesis. Especially for mutagenizing
large peptide additions, it may be relevant to use PCR generated mutagenesis (e.g. as
described by Deshler 1992 or Leung et aL, 1989), in which one or more suitable

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oligonucleotide probes are used which flanks the area to be mutagenized. For mutagenesis of
shorter peptide additions, it is more preferably perform the localized random mutagenesis by
use of doped or spiked oligonucleotides. The doping or spiking is used, e.g., to avoid codons
for unwanted amino acid residues or to increase the likelihood that a particular type of amino
5 acid residue, such as a positively charged or hydrophobic amino acid residue, is introduced at
a desired position.

Subsequent to the mutagenesis the mutated DNA is expressed by culturing a suitable host
cell carrying the DNA sequence under conditions allowing expression to take place/The host
cell used for this purpose may be one which has been transformed with the mutated DNA
10 sequence, optionaly present on a vector, or one which carried the DNA sequence encoding the
parent enzyme during the mutagenesis treatment. Examples of suitable host cells are given
below, and is preferably a host cell which is capable of secreting the mutated enzyme
(enabling an easy screening). Yeast cells, such as cells of 5. cereviciae, have been found to be
suitable host cells.

15 The screening criteria of step c) will have to be chosen in dependence of the desired
properties of the modified lipolytic enzyme. If it is desirable to construct a modified lipolytic
enzyme with an improved wash performance the screening is conveniently conducted for a
reduced dependency to calcium and/or an improved tolerance towards a detergent or a
detergent component. The detergent or detergent component may be any of the specific

20 components mentioned further below in the Detergent Composition section. A preferred
detergent component is a non-ionic or an anionic surfactant such as an alcohol ethoxylate or
LAS, a preferred detergent is the detergent PCS described in the Materials and methods
section below. Non-ionic surfactants are of particular interest for screening of H. lanuginosa
type of lipases (e.g. fungal lipases) whereas an-ionic surfactants are of interest for screening

25 of Pseudomonas type lipases.

The screening of step c) is coneveniently performed by use of a filter assay based on the
following principle:

A microorganism capable of expressing the modified lipolytic enzyme of interest is
incubated on a suitable medium and under suitable conditions for the enzyme to be secreted,
30 the medium being provided with a double filter comprising a first protein-binding filter and on
top of that a second filter exhibiting a low protein binding capability. The microorganism is
located on the second filter. Subsequent to the incubation, the first filter comprising enzymes
secreted from the microorganisms is separated from the second filter comprising the micro-

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organisms. The first filter is subjected to screening for the desired enzymatic activity and the
corresponding microbial colonies present cm the second filter are identified.

The filter used for binding the enzymatic activity may be any protein binding filter e.g.
nylon or nitrocellulose. The topfilter carrying the colonies of the expression organism may be
5 any filter that has no or low affinity for binding proteins e.g. cellulose acetate or Durapore™.
The filter may be pretreated with any of the conditions to be used for screening or may be
treated during the detection of enzymatic activity.

The enzymatic activity may be detected by a dye, flourescence, precipitation, pH indicator,
IR-absorbance or any other known technique for detection of enzymatic activity,
to Hie detecting compound may be immobilized by any immobilizing agent e.g. agarose,
agar, gelatine, polyacrylamide, starch, filter paper, cloth; or any combination of immobilizing
agents.

Lipase activity may be detected by Brilliant green, Rhodamine B or Sudan Black in
combination with a lipid e.g. olive oil or laid. The screening criteria for identifying modified

15 lipolytic enzymes having improved washing performance may be e.g. EGTA, EDTA, non-
ionic or anionic tensides, alkaline pH, or any detergent composition in combination with one
of the above detectors of enzymatic activity.

It will be understood that the screening criteria used in the filter assay of the invention may
be chosen so as to comply with the desired properties or uses of the enzymes to be screened.

20 For instance, in a screening for lipolytic enzymes of particular use in the paper and pulp
industry, it may be relevant to screen for an acid enzyme having an increased temperature sta-
bility. This may be performed by using a buffer with acidic pH (e.g. pH 4) and/or incubate
under higher temperature before or under the assay. For detergent enzymes screening is
normally conducted at alkaline pH.

25 Alternatively, the screening may be performed by isolating the mutated lipolytic enzyme
resulting from step b) and testing the wash performance (or any other relevant property)
thereof. Also, the latter "in vivo" test may be used in addition to die screening assay so as to
identify the best of the mutated lipolytic enzymes selected in the screening assay. Finally,
amino acid sequencing of the resulting modified lipolytic enzyme may be used to confirm the

30 amino acid sequence of the peptide addition.

Method of improving properties

It is also an object of the invention to improve properties, in particular to improve the wad)

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performance, of the parent lipolytic enzymes mentioned above. The improved properties
obtained by to the method of the invention is believed to be a result of an increased affinity
towards the lipid substrate.

The method of the invention comprises applying a peptide addition to the N-terminus or C-
5 terminus of the parent enzyme in its mature form. In an embodiment of the invention this is
done by applying a peptide addition to the parent enzyme by cultivating a host cell comprising
a DNA sequence encoding the pre, pro or prepro-form of the parent lipolytic enzyme.
Optionally the DNA sequence is present on a vector. The recovering of the resulting
modified lipolytic enzyme, the host cell, cultivation conditions and/or recovery conditions are

10 selected so that at the most a partial processing of the pre, pro or prepro-form of the parent
enzyme occur resulting in that at least 5%, such as at least 10%, such as at least 15%, such
as at least 20% , such as at least 25%, such as at least 50% , such as at least 75% of the
produced modified enzyme molecules comprise the desired peptide addition, e.g. the entire
pro-sequence or a substantial part thereof.

is The host cell may be of a different origin than the parent enzyme, e.g. of another genus
than the one from which the parent enzyme is derived, or may have another posttranslational
processing machinery than the source of the parent enzyme.

The parent lipolytic enzyme is preferably derived from a filamentous fungus, such as a
strain of a Humicola sp., in particular H. lanuginosa, or a bacterium, such as a strain of a

20 Pseudomonas sp., and the host cell is a yeast celll, such as a strain of Sacchawmyces sp., in
particular Sacchawmyces cerevisiae, or a strain of Hansenula sp..

In an embodiment of the invention the inherent proteolytic enzyme producing capability of
the host cell used for applying the peptide addition to the parent lipolytic enzyme has been
reduced, e.g. by abolishing the production of one or more proteolytic enzymes by the host

25 cdl.

The method of the invention comprises the steps of:

a) subjecting a DNA sequence encoding the parent lipolytic enzyme with a peptide addition,
such as a DNA sequence of the invention, to localized random mutagenesis in the part of
the DNA sequence encoding the peptide addition or a non-structural part of the C-terminal

30 or N-terminal end of the parent enzyme,

b) expressing the mutated DNA sequence obtained in step a) in a host cell, and

c) screening for host cells expressing a mutated lipolytic enzyme which has an improved
performance as compared to the parent lipolytic enzyme.

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In an embodiment the DNA sequence is the gene or cDNA sequence encoding the parent
enzyme in its pro or prepro-form.

It is also contemplated to according to the invention to introduce a mutation in the non-
structural part of the C-terminus or N-terminus of the parent enzyme in its mature form, e.g.
5 by deleting or replacing a negatively charged amino acid residue of the non-structural part
with a neutral or positively charged amino acid residue or with a hydrophobic amino arid
residue, or replacing a neutral amino acid residue with a positively charged amino arid
residue.

10 Parent lipolytic enzyme

The modified lipolytic enzymes of the invention may be constructed from any parent
lipolytic enzymes of microbial origin, preferably of bacterial or fungal (i.e. filamentous fungal
or yeast) origin.

According to the invention the enzyme of the invention may be any lipolytic enzyme
15 including lipases, phospholipases, esterases and cutinases (according to conventional
terminology).

It is to be understood that lipolytic enzymes normally comprising pro- and/or pre-peptides
in their unprocessed state as well as enzymes which do not are contemplated to serve as parent
enzymes for the modification according to the invention.
20 Examples of relevant parent lipolytic enzymes include enzymes derived from die
following microorganisms:

Humicola, e.g. ft brevispora, ft lanuginosa, H. brevis var. thermoidea and ft insolens

(US 4,810,414) or WO 96/13580;

PseudomonaSy e.g. ft. fiagU ft. stutzeri, ft. cepacia and ft. fluorescent (WO
25 89/04361), or ft. ptantarii or ft. gladioli (US patent no. 4,950,417 (Solvay enzymes))
or ft. alcaligenes and ft. pseudoalcaligenes (EP 218 272 or WO 94/25578) or ft.
mendoana (WO 88/09367; US 5,389,536);

Fusarium, e.g. F. axysporwn (EP 130,064) or F. solampisi (WO 90/09446);
Mucor (also called Rhizomucor), e.g. M. miehei (EP 238 023);
30 Absidia sp. (WO 96/13578);

Chromobaaerium (especially C. viscosum);
Aspergillus (especially A. mger)\

Candida, e.g. C. cylindracea (also called C rugosa) or C. amaraica (WO 88/02775) or

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C. amaraica lipase A or B (WO 94/01541 and WO 89/02916);
Geotricum, e.g. G. cartdidum (Schimada et ah, (1989), J.Biochem., 106, 383-388);
PemcUtium, e.g. P. camembertii (Yamaguchi el al.,(1991), Gene 103, 61-67);
Rhizopus, e.g. R. delemar (Hass et aL, (1991), Gene 109,107-113) or R. niveus
(Kugimiya et aL, (1992) Biosci.Biotech. Biochem 56, 716-719) or R. oryzae; and
Bacillus, e.g. B. subtilis (Dartois et al., (1993) Biochemica et Biophysica acta 1131,
253-260) or B. stearothermophilus (IP 64/7744992) or B. pumilus (W091/16422).
The generic name Thermomyces lanuginosus was introduced by Tsiklinsky in 1899
for one species, Thermomyces lanuginosus. The species is very characteristic
morphologically and physiologically and there is little doubt that Tsiklinsky's
Thermomyces lanuginosus is the same as isolated and described by later workers (such as
for instance Bunce (1961) and Cooney & Emerson (1964) as Humicola lanuginosa.

According to The International Code of Botanical Nomenclature the correct name
is the earliest legitimate one. Thus Thermomyces lanuginosus should be the correct name.
However, due to Tsiklinsky's incomplete description of the species, this legitimacy has
been questioned by many mycologists. Thus the main reason for the taxonomical .
confusion relates to different views as to whether Tsiklinsky's description meets the
requirements for a valid publication.

However, the name Humicola lanuginosa is still seen in literature, probably due
to the popularity of the book of Cooney & Emerson (1964) "The Thermophilic Fungi",
which recommends the use of the name Humicola lanuginosa.

Sequencing of the DNA encoding part of the 18S ribosomal gene from
Thermomyces lanuginosus have been performed. The 18S sequences was compared to
other 18S sequences in the GenBank database and a phylogenetic analysis using
parsimony (PAUP, Version3.1.1, Smithsonian Institution, 1993) have also been made.
This clearly assigns Thermomyces lanuginosus to the class of Plectomycetes, probably to
the order of Euronales. According to the Entrez Browser at the NCBI (National Center
for Biotechnology Information), this relates Thermomyces lanuginosus to families like
Eremascaceae, Monoascaceae, Pseudoeurotiaceae and Trichocomaceae, the latter
containing genera like Emericella 9 Aspergillus, Penicillium f Eupenicillium 9 Paecilomyces,

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Talaromyces, Thermoascus and Sclerocleista.

In connection with the present invention the name Hwnicola lanuginosa will be used.
In connection with the Pseudomonas sp. lipases it has been found that lipases from the

following organisms have a high degree of homology and thus are contemplated to belong to
5 the same family of lipases: ft. ATCC21808, ft. aeruginosa EF2, ft. aeruginosa PAC1R,

ft. aeruginosa PAOl, ft. aeruginosa TE3285, ft. sp. 109, ft. pseudoalcaligenes Ml, Ps.

glumae, ft. cepacia DSM3959, ft. cepacia M-12-33, ft. sp. KWI-56, ft. purida IF03458,

ft. putida IFO12049 (Gilbert, E. J., (1993), Pseudomonas lipases: Biochemical properties

and molecular cloning. Enzyme Microb. TechnoL, 15, 634-645).
10 Specific examples of readily available commercial lipases which may be modified

according to the invention include Lipolase® and Lipolase® Ultra (available from Novo

NordiskA/S).

Examples of other lipolytic enzymes specifically contemplated according to the invention
are Lumafast® (a ft. mendocina lipase from Genencor Int. Inc); lipomax® (a ft.
15 pseudoalcaligenes lipase from Genencor Int. Inc.); a Fusariwn solani lipase (cutinase) from
Unilever; a Bacillus sp. lipase from Solvay enzymes; and Liposam®, (a ft. mendocina lipase
from Showa Denko) and further the Peudomonas sp. lipase described in WO 95/06720 which
have been sequenced and found to have the amino acid sequence shown in SEQ ID NO. 93.

It is to be emphasized that the parent lipolytic enzyme from which the modified ehzyme
20 of the invention can be constructed includes the above mentioned lipolytic enzymes and any
variant, modification, and truncation thereof.

Examples of such parent enzymes which are specifically contemplated include the
enzymes described in WO92/05249, WO 94/01541, WO 94/14951, WO 94/25577, WO
95/22615 and a protein engineered lipase variants as described in EP 407 225; a protein
25 engineered ft. mendocina lipase as described in US 5,352,594; a cutinase variant as described
in WO 94/14964; a variant of an Aspergillus lipolytic enzyme as described in EP patent
167,309; and Peudomonas sp. lipase described in WO 95/06720.

In the most preferred embodiment die parent enzyme is derived from a strain of a
Humicola sp. or or from a strain of a Pseudomonas sp. or a genus considered to belong to the
30 Pseudomonas family.

In a specific embodiment of the invention the DNA sequence encoding the parent enzyme
with lipolytic activity (to be processed into a modified enzyme of the invention) is the DNA
sequence encoding the enzyme with lipolytic activity derived from the filamentous fungi

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Humicola lanuginosa described in EP 305 216. The amino acid sequence of the parent
enzyme is in this case that of the secreted mature enzyme.

It is presently contemplated that the washing performance and/or thermostability of the
modified enzyme of the invention may be further improved if the enzyme is glycosylated.
Accordingly, in an embodiment of the invention the modified enzyme may be glycosylated.
The amino acid sequence may have any degree of glycosylation.

DNA sequence of the invention

In another aspect the invention relates to a DNA sequence encoding said modified enzyme
with lipolytic activity of the invention.

The DNA sequence encoding a modified enzyme of the invention is normally prepared by
modification of the DNA sequence encoding the parent lipolytic enzyme in question by any
suitable method known in the art(e.g. by use of PCR as mentioned above), but may also be
prq«red synthetically by established standard methods, e.g. the phosphoamidite method
described by Beaucage and Caruthers, (1981), Tetrahedron Letters 22, 1859 - 1869, or the
method described by Matthes et al., (1984), EMBO Journal 3, 801-805. According to the
phog>hoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA
synthesizer, purified, annealed, ligated and cloned in suitable vectors.

A DNA sequence encoding the parent lipolytic enzyme of choice may be obtained by use
of conventional techniques. For instance, the DNA sequence may be isolated from a genomic
or DNA library prepared from the relevant organism or may be obtained by expression
cloning, e.g. as described in WO 93/1 1249, or may be produced synthetically.

In a currently preferred embodiment, the DNA sequence encoding the modified lipolytic
enzyme of the invention is prepared from the DNA sequence encoding a lipolytic enzyme
derived from Humicola lanuginosa and described in EP 305 216. In another preferred
embodiment the DNA sequence is derived from a strain of Pseudomonas sp. 9 especially a
strain of ft. alcaligenes or ft. pseudoalcaligenes.

An isolated nucleic arid sequence encoding a modified lipolytic enzyme of the invention
may be manipulated in a variety of ways to provide for expression of the enzyme. Manipulation
of the nucleic arid sequence encoding a modified lipolytic enzyme prior to its insertion into a
vector may be desirable or necessary depending on the expression vector. The techniques for
modifying nucleic arid sequences utilizing cloning methods are well known in the art.

The term "control sequences" is defined herein to include all components which are

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necessary or advantageous for expression of the coding sequence of the nucleic acid sequence.
Each control sequence may be native or foreign to the nucleic acid sequence encoding the
modified lipolytic enzyme. Such control sequences include, but are not limited to, a leader, a
polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a
transcription terminator. At a minimum, the control sequences include a promoter, and
transcriptional and translational stop signals. The control sequences may be provided with linkers
for the purpose of introducing specific restriction sites facilitating ligation of the control
sequences with the coding region of the nucleic acid sequence encoding a modified lipolytic
enzyme.

The control sequence may be an appropriate promoter sequence, a nucleic acid sequence
which is recognized by a host cell for expression of the nucleic acid sequence. The promoter
sequence contains transcription and translation control sequences which mediate the expression
of the modified lipolytic enzyme. The promoter may be any nucleic add sequence which shows
transcriptional activity in the host cell of choice and may be obtained from genes encoding
extracellular or intracellular polypeptides other homologous or heterologous to the host cdL
Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the
present invention, especially in a bacterial host cell, are the promoters obtained from the £ coli
lac operon, the Streptomyces coelicolor agarase gene (dagA\ the A subtilis levansucrase gene
(sacB) or the alkaline protease gene, the B. licheniformis alpha-amyiase gene (amyL), the B.
stearothermophilus mahogenic amylase gene {amyM), the 2JL amyloliquefaciens alpha-amylase
gene (amyQ\ the BMchemformis penicillinase gene (penP), the & subtilis xylA and xylB genes,
the B. pumibis xylosidase gene, and the prokaryotic beta-lactamase or tryptophan gene (ViDa-
Kamaroff et aL, 1978, ProceeOngs of the National Academy of Sciences USA 75:3727-373 1), as
well as the toe gene (DeBoer et oi, 1983, ProceecSngs of the National Academy of Sciences
USA 80:21-25). Further promoters are described in Useful proteins from recombinant bacteria"
in Scientific American, 1980, 242:74-94; and in Sambrook et aL, 1989, supra. Examples of
suitable promoters for directing the transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host ceil are promoters obtained from die genes encoding A
oryzae TAKA amylase, A. oryzae triose phosphate isomerase, Rhizomucor miehei asparttc
proteinase, A. niger neutral alpha-amylase, A. niger add stable alpha-amylase, A. niger or
A„awamori gtucoamylase (glaA) 7 Rhizomucor miehei lipase, A. oryzae alkaline protease, A.
oryzae triose phosphate isomerase, A. mdulans acetamidase, Fusahum oxysporum trypsin-like
protease (as described in U.S. Patent No. 4,288,627, which is incorporated herein by referenceX

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or the ADH-3 promoter (McKnight et al., (1985), The EMBO J. 4, 2093-3099) and hybrids
thereof Particularly preferred promoters for use in filamentous fungal host cells is the TAKA
amylase and the glaA promoters. In a yeast host, promoters from yeast glycolytic genes
(Hitzeman et al.,(1980), J. Biol. Chem. 255, 12073-12080; Alber and Kawasaki, (1982X J. Mol.
5 Appl. Gen. 1, 419-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of
Microorganisms for Chemicals (HoDaender et al, eds), Plenum Press, New York, 1982), or the
TPI1 (US 4,599,311) or ADH2^c (Russell et al., (1983), Nature 304, 652-654) promoters,
useful promoters are obtained from the £ cerevisiae enolase (ENO-1) gene, the £ cerevisiae
galactokinase gene (GAL1), the £ cerevisiae alcohol dehydrogenase/glyceraldehyde-3-pho^)hate
to dehydrogenase genes (ADH2/GAP), and the X cerevisiae 3-phosphoglycerate kinase gene.
Other useful promoters for yeast host cells are described by Romanos et aL, 1992, Yeast 8:423-
488.

The control sequence may also be a suitable transcription terminator sequence, a
sequence recognized by a host cell to terminate transcription. The terminator sequence is

15 operably linked to the 3' terminus of the nucleic acid sequence encoding the modified lipolytic
enzyme. The terminator sequence may be native to the nucleic add sequence encoding the
modified lipolytic enzyme or may be obtained from foreign sources. Any terminator winch is
functional in the host cell of choice may be used in the present inventioa Preferred terminators
for filamentous fungal host cells are obtained from the genes encoding A. oryzae TAKA amylase,

20 A. niger glucoamylase, A. nidulans anthrairilate synthase, A. niger alpha-ghicosidase, and
Fusarium oxysporum trypstn-like protease. Preferred terminators for yeast host cells are obtained
from the genes encoding £ cerevisiae enolase, £ cerevisiae cytochrome C (CYC IX or £
cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host
ceDs are described by Romanos etaL, 1992, supra

25 The control sequence may also be a suitable leader sequence, a nontranslated region of a

mRNA which is important for translation by the host ceD. The leader sequence is operably linked
to the 5' terminus of the nucleic acid sequence encoding the modified lipolytic enzyme. The
leader sequence may be native to the nucleic acid sequence encoding the modified lipolytic
enzyme or may be obtained from foreign sources. Any leader sequence which is functional in the

30 host ceD of choice may be used in the present inventioa Preferred leaders for filamentous fungal
host cells are obtained from the genes encoding A. oryzae TAKA amylase and A. oryzae triose
phosphate isomerase. Suitable leaders for yeast host cells are obtained from the £ cerevisiae
enolase (ENO-1) gene, the £ cerevisiae 3-phosphoglycerate kinase gene, the £ cerevisiae alpha-

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6ctor, and the S. cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
genes (ADH2/GAP).

The control sequence may also be a potyadenyiarion sequence, a sequence which is
operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is
recognized by the host cefl as a signal to add polyadenosine residues to transcribed mRNA. The
polyadenylation sequence may be native to the nucleic acid sequence encoding the modified
lipolytic enzyme or may be obtained from foreign sources. Any polyadenylation sequence which
is functional in the host cell of choice may be used in the present invention. Preferred
polyadenylation sequences for filamentous fungal host cells arc obtained from the genes encoding
A. oryzae TAKA amylase, A. niger glucoamyiase, A. nidulans anthranilate synthase, and A. niger
alpha-ghicosidase. Useful polyadenylation sequences for yeast host cells are described by Guo
and Sherman, 1995, Molecular Cellular Biology 15:5983-5990. Polyadenylation sequences are
weD known in the art for mammalian host cells.

The control sequence may also be a signal peptide coding region, which codes for an
amino acid sequence linked to the amino terminus of the modified lipolytic enzyme which can
direct the expressed modified lipolytic enzyme into the cell's secretory pathway. The signal
peptide coding region may be native to the modified lipolytic enzyme of the invention or may be
obtained from foreign sources. The 5* end of the coding sequence of the nucleic add sequence
may inherently contain a signal peptide coding region naturally linked in translation reading frame
with the segment of the coding region which encodes the secreted modified lipolytic enzyme.
Alternatively, the 5* end of the coding sequence may contain a signal peptide coding region which
is foreign to that portion of the coding sequence which encodes the seemed modified lipolytic
enzyme. The foreign signal peptide coding region nay be required where the coding sequence
does not normally contain a signal peptide coding region. Alternatively, the foreign signal peptide
coding region may amply replace the natural signal peptide coding region in order to obtain
enhanced secretion of the enzyme relative to the natural signal peptide coding region normally
associated with the coding sequence. The signal peptide coding region may be obtained from a
ghicoamylase or an amylase gene from an Aspergillus species, a lipase or proteinase gene from a
Rhizomucor species, the gene for the a-factor from Saccharomyces cerevisiae, an amylase or a
protease gene from a Bacillus species, or the calf preprochymosin gene. An effective signal
peptide coding region for bacterial host cells is the signal peptide coding region obtained from the
mahogerric amylase gene from Bacillus NCIB 1 1837, the B. stearothermophitus alpha-amylase
gene, the B. Ucheniformis subtifisin gene, the B. licheniformis beta-lactamase gene, the A

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stearothermophitus neutral proteases genes (nprT, nprS, nprM), and the B. subtilis PrsA gene.
Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews
57:109-137. An effective signal peptide coding region for filamentous fungal host cells is the
signal peptide coding region obtained from A. oryzae TAKA amylase gene, A. mger neutral
5 amylase gene, the Rhizomucor miehei aspartic proteinase gene, the H. lanuginosa ceDulase gene,
or the Rhizomucor miehei lipase gene. Useful signal peptides for yeast host cells are obtained
from the genes for S. cerevisiae a-factor and S. cerevisiae invertase. Other useful signal peptide
coding regions are described by Romanos et at, 1992, supra. However, any signal peptide
coding region capable of directing the expressed enzyme into the secretory pathway of a host cell

to of choice may be used in the present invention

The nucleic acid constructs of the present invention may also comprise one or more
nucleic acid sequences which encode one or more factors that are advantageous in the expression
of the modified liporytic enzyme, e.g, an activator (e.g., a trans-acting factor), a chaperone, and a
processing protease. The nucleic acids encoding one or more of these factors are not necessarily

15 in tandem with the nucleic acid sequence encoding the modified lipolytic enzyme. An activator is
a protein which activates transcription of a nucleic acid sequence encoding a modified lipolytic
enzyme (Kudla et aL, 1990, EMBO Journal 9:1355-1364; Jarai and Buxton, 1994, Current
Genetics 26:2238-244; Verdier, 1990, Yeast 6:271-297). The nucleic acid sequence encoding an
activator may be obtained from the genes encoding B. stearothermophilus NprA (rqxA), S.

20 cerevisiae heme activator protein 1 (hap J), S cerevisiae galactose metabolizing protein 4 (gal4),
and A. rudulans ammonia regulation protein (areA). For further examples, see Verdier, 1990,
supra and MacKenzie et aL, 1993, Journal of General Microbiology 1392295-2307. A
chaperone is a protein which assists another polypeptide in folding properly (Haiti et a!., 1994,
UBS 1920-25; Bergeron et aL, 1994, TIBS 19:124-128; Demolder et aL, 1994, Journal of

25 Biotechnology 32:179-189; Craig, 1993, Science 260:1902-1903; Gething and Sambrook, 1992,
Nature 35533-45; Puig and Gilbert, 1994, Journal of Biological Chemistry 269:7764-7771;
Wang and Tsou, 1993, 77k FASEB Journal 7:1515-11157; Robinson et aL, 1994,
Bio/Technology 1381-384). The nucleic acid sequence encoding a chaperone may be obtained
from the genes encoding B. subtilis GroE proteins, A. oryzae protein disulphide isomerase, S

30 cerevisiae calnexin, 5 cerevisiae BDVGRP78, and S cerevisiae Hsp70. For further examples,
see Gething and Sambrook, 1992, supra, and HartI et al., 1994, supra. Any factor that is
functional in the host cell of choice may be used in the present invention.

It may also be desirable to add regulatory sequences which allow the regulation of the

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expression of the modified lipolytic enzyme relative to the growth of the host cell. Examples of
regulatory systems are those which cause the expression of the gene to be turned on or off in
response to a chemical or physical stimulus, including the presence of a regulatory compound.
Regulatory systems in prokaryotic systems would include the lac, tac, and trp operator systems.

5 In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-
amylase promoter, A. niger glucoamylase promoter, and the A. oryzae glucoamylase promoter
may be used as regulatory sequences. Other examples of regulatory sequences are those which
allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase
gene which is amplified in the presence of methotrexate, and the metallothionein genes which are

10 amplified with heavy metals. In these cases, the nucleic add sequence encoding the modified
lipolytic enzyme would be placed in tandem with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a nucleic

15 acid sequence of the present invention, a promoter, and transcriptional and translational stop
signals. The various nucleic acid and control sequences described above may be joined together
to produce a recombinant expression vector which may include one or more convenient
restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the
modified lipolytic enzyme at such sites. Alternatively, the nucleic add sequence of the present

20 invention may be expressed by inserting the nudeic add sequence or a nuddc add construct
comprising the sequence into an appropriate vector for expression. In creating the expression
vector, the coding sequence is located in the vector so that the coding sequence is operably linked
with the appropriate control sequences for expression, and possibly secretion.

The recombinant expression vector may be any vector which can be conveniently

25 subjected to recombinant DNA procedures and can bring about the expression of the nuddc add
sequence. The choice of the vector will typically depend on the compatibility of the vector with
the host cell into which the vector is to be introduced. The vectors may be linear or dosed
circular plasmids. The vector may be an autonomously replicating vector, i.e., a vector which
exists as an extrachromosomal entity, the replication of which is independent of chromosomal

30 replication, e.g., a plasmid, an extrachromosomal dement, a minichromosome, or an artificial
chromosome. The vector may contain any means for assuring self-replication. Alternatively, the
vector may be one which, when introduced into the host cell, is integrated into the genome and
replicated together with the chromosome(s) into which h has been integrated. The vector system

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may be a single vector or plasmid or two or more vectors or plasmids which together contain the
total DNA to be introduced into the genome of the host cell, or a transposoa

The vectors of the present invention preferably contain one or more selectable markers
which permit easy selection of transformed cells. A selectable marker is a gene the product of
which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to
auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from B.
subtitis or B. ticheniformis, or markers which confer antibiotic resistance such as ampicillin,
kanamycin, chloramphenicol or tetracycline resistance. A frequently used mammalian marker is
the dihydrofolate reductase gene. Suitable markers for yeast host cells are ADE2, fflS3, LEU2,
LYS2, MET3, TOPI, and URA3. A selectable marker for use in a filamentous fungal host cell
may be selected from the group including, but not limited to, amdS (acetamidase), argB
(ornithine carbamoyhransferase), bar (phosphinothricin acetyttransferase), hygB (hygromycin
phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-S'-phosphate decaiboxylase), sC
(sulfate adenykransferase), trpC (anthranilate synthase), and glufosinate resistance markers, as
well as equivalents from other species. Preferred for use in an Aspergillus ceB are the amdS and
pyrG markers of A. mdulcms ox A. oryzae and the bar marker of Streptomyces hygrosccpicus.
Furthermore, selection may be accomplished by co-transformation, e.g., as described in WO
91/1 7243, where the selectable marker is on a separate vector.

The vectors of the presort invention preferably contain an elements) that permit* stable
integration of the vector into the host cell genome or autonomous replication of the vector in the
cell independent of the genome of the cell.

The vectors of the present invention may be integrated into the host cell genome when
introduced into a host cell. For integration, the vector may rely on the nucleic acid sequence
encoding the modified lipolytic enzyme or any other dement of the vector for stable integration
of the vector into the genome by homologous or nonhomologous recombination. Alternatively,
the vector may contain additional nucleic add sequences for directing integration by homologous
recombination into the genome of the host cell. The additional nucleic acid sequences enable the
vector to be integrated into the host cell genome at a precise locations) in the chromosomes).
To increase the likelihood of integration at a precise location, the integrational dements should
preferably contain a suffident number of nuddc adds, such as 100 to 1,500 base pairs, preferably
400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly
homologous with the corresponding target sequence to enhance the probability of homologous
recombination. The integrational dements may be any sequence that is homologous with the

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target sequence in the genome of the host cell. Furthermore, the integrational dements may be
non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be
integrated into the genome of the host cell by non-homologous recombination. These nucleic acid
sequences may be any sequence that is homologous with a target sequence in the genome of the
host cell, and, furthermore, may be non-encoding or encoding sequences.

For autonomous replication, the vector may further comprise an origin of replication
enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial
origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177,
pACYCI84, pUBl 10, pE194, pTA1060, and pAMBl. Examples of origin of replications for use
in a yeast host cell are the 2 micron origin of replication, the combination of CEN6 and ARS4,
and the combination of CEN3 and ARS1 . The origin of replication may be one having a mutation
which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehriich, 1978,
Proceedings of the National Academy of Sciences USA 75: 1433).

More than one copy of a nucleic add sequence encoding a modified lipolytic enzyme of
the present invention may be inserted into the host cell to amplify expression of the nucleic acid
sequence. Stable amplification of the nucleic acid sequence can be obtained by integrating at least
one additional copy of the sequence into the host cell genome using methods well known in the
art and selecting for transformants.

The procedures used to ligate the elements described above to construct the recombinant
expression vectors of the present invention are well known to one skilled in the art (see, e.g. y
Sambrook etaL, 1989, supra).

Host Cells

The present invention also relates to recombinant host cells, comprising a nucleic acid
sequence of the invention, which are advantageously used in the recombinant production of the
modified lipolytic enzymes. The cell is preferably transformed with a vector comprising a nucleic
acid sequence of the invention followed by integration of the vector into the host chromosome.
"Transformation" means introducing a vector comprising a nucleic acid sequence of the present
invention into a host ceD so that the vector is maintained as a chromosomal integrant or as a self-
replicating extra-chromosomal vector. Integration is generally considered to be an advantage as
the nucleic acid sequence is more Ekdy to be stably maintained in the cdL Integration of the
vector into the host chromosome may occur by homologous or non-homologous recombination
as described above.

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The choice of a host cell will to a large extent depend upon the gene encoding the
modified lipolytic enzyme and its source. In addition, the choice of host cell will often depend on
the proteolytic enzyme system of the host cell and its impact on the production of a modified
lipolytic enzyme of the invention. Accordingly, it may be desirable to use a host cell which is
deficient in one or more proteolytic enzymes or other enzyme processing means. Protease
deficient host cells of bacteria as well as fungal (filamentous fungal and yeast) cells are well-
known in the art.

The host cell may be a unicellular microorganism or a non-unicellular microorganism.
Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited
to, a Bacillus cell, eg, B. subtilis, B. licheniformis y B. lentos, B. brevis, B. stearothermophihis,
R alknlophihis, B. amyloliquefaciens, B. coagulans, B.tirculans, B. lautus, B. megaterium, and
B. thuringiensisr, or a Streptomyces cell, e.&, £ Ihidans or £ murinus, or gram negative bacteria
such as K colt and Pseudomohas sp. (especially when a bacterial lipolytic enzyme, such as a
Pseudomonas sp. enzyme is to be produced). The transformation of a bacterial host cell may, for
instance, be effected by protoplast transformation (see, e.g. y Chang and Cohen, 1979, Molecular
General Genetics 168:111-115), by using competent cells (see, e.g., Young and Spizizin, 1961,
Journal of Bacteriology 81:823-829, or Dubnar and Davidoff-Abelson, 1971, Journal of
Molecular Biology 56209-221X by electroporation (see, e.#, Shigekawa and Dower, 1988,
Biotechniques 6:742-751), or by conjugation (see, e.&, Koehler and Thome, 1987, Journal of
Bacteriology 169:5771-5278).

The host cefl may be a eukaiyote, and is preferably a fungal, i.e. a yeast cell or a
filamentous fungal cell, especially for the production of a modified lipolytic enzyme of eukaiyotic
origin.

"Yeast" as used herein includes ascosporogenous yeast (EndomycetalesX
basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The
ascosporogenous yeasts are divided into the families Spermophthoraceae and
Saccharomycetaceae. The latter is comprised of four subfamilies, Schtzosaccharomycoideae (eg,
genus Schhasaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (eg.,
genera Pichia, Khiyveromyces and Saccharomyces). The basidiosporogenous yeasts include the
genera Leucospohdim y Rhodosporidhim, SporicSobohis, Filobasidhtm, and Filobasidiella.
Yeast belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae
{e g, genera Sorobolomyces and BuUera) and Cryptococcaceae (e.g. , genus Candida). Since the
classification of yeast may change in the future, for the purposes of this invention, yeast shall be

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defined as described in Biology ami Activities of Yeast (Skinner, F.A., Passmore, S.M., and
Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980. The biology of yeast
and manipulation of yeast genetics are wefl known in the art (see, e.g y Biochemistry and
Genetics of Yeast, Bacil, M, Horecker, B.J., and Stopani, A.OM, editors, 2nd edition, 1987;
77k? Yeasts, Rose, AH, and Harrison, IS., editors, 2nd edition, 1987; and The Molecular
Biofogy of the Yeast Saccharomyces, Strathem et at, editors, 1981). In connection with the
present invention the use of yeast cells which typically have another proteolytic enzyme
processing system that, e.g., bacteria and filamentous fungi, may be of particular use for
preparing modified lipolytic enzymes which, as the peptide addition, comprise a part or all of the
natural prosequences of the parent lipolytic enzyme in question. When the fungal tost cell is a
yeast cell (e.g. to be used in applying a peptide addition (in the form of part of or the entire
prosequence of the parent enzyme, the yeast host cell may be a cell of a species of Cam£da,
Khiyveromyces, Saccharomyces, Schizosaccharomyces, Pichia, or Yarrowia, such as a £
cerevkdae cell, zZscarlsbergensis, a£ (Bastaticus cell, a S. douglasii cell, a £ khryveri cell, a£
norbensis cell, or a £ cwiformis cell.

"Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytricfiomycota,
and Zygomycota (as defined by Hawksworth et aL, In, Ainsworth andBisby f s Dictionary of The
Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the
Oomycota (as cited in Hawksworth et aL, 1995, supra, page 171) and all mhosporio fungi
(Hawksworth et aL, 1995, supra\ Representative groups of Ascomycota include, e.g.,
Neuraspora, EupeniciUhtm (=PemciUhun), Emericella (^Aspergillus), Eurothmt (^Aspergillus),
and the true yeasts listed above. Examples of Basidiomycota include mushrooms, rusts, and
smuts. Representative groups of Chytridiomycota include, e.g, Allomyces, BlastoclacBeUa,
Coelomomyces, and aquatic fungi. Representative groups of Oomycota include, e.g.,
Saprolegroomycetous aquatic fungi (water molds) such as Achfya. Examples of mkosporic fungi
include Aspergillus, Penicillium, Candida, and Alternaria. Representative groups of
Zygomycota include, e.g, Rhizcpus arid Mucor.

"Filamentous fungi" include aO filamentous forms of the subdivision Eumycota and
Oomycota (as defined by Hawksworth et a/., 1995, supra). The filamentous fungi are
characterized by a vegetative mycelium composed of chitin, cellulose, gfucan, chitosan, mannan,
and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon
catabolism is obligatdy aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces
cerevisiae is by budding of a unicellular thalhis and carbon catabolism may be fermentative.

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In a preferred embodiment, the fungal host cell is a filamentous fungal cell. In a more
preferred embodiment, the filamentous fungal host cell is a cell of a species o£ but not limited to,
Acremonium, Aspergillus, Fusarium, Humicola y Myceliophthora y Mucor, Neurospora,
Penicillhim, Thielavia, Totypocladhan, and Trichoderma. In an even more preferred
embodiment, the filamentous fungal host cell is an Aspergillus cell. In another even more
preferred embodiment, the filamentous fungal host cell is a Fusarium ceD. In a most preferred
embodiment, the filamentous fungal host cell is an A oryzae cell, an A niger cell, an A foetidus
cell, or an A.japomcus cell. In another most preferred embodiment, the filamentous fungal host
cell is a Fusarium oxysporum cell or aF. grammearum cell.

Fungal cells may be transformed by a process involving protoplast formation,
transformation of the protoplasts, and regeneration of the cell wall in a manner known per se.
Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and
Yekon et aL, 1984, Proceedings of the National Academy of Sciences USA 81>1470-1474. A
suitable method of transforming Fusarium species is described by Malardier et al. y 1989, Gene
78:147-156 or in WO 96/00787. Yeast may be transformed using the procedures described by
Becker and Guarente, In Abdson, IN. and Simon, ML, editors, Guide to Yeast Genetics and
Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc.,
New York; Ito et al y 1983, Journal of Bacteriology 153:163; and Hnnen et aL % 1978,
Proceedings of the National Academy of Sciences USA 75:1920. Mammalian cells may be
transformed by direct uptake using the calcium phosphate precipitation method of Graham and
Van der Eb (1978, Virology 52:546).

Host cells used according to the invention may advantagously be protease deficient or
protease minus strains (or the like), such as strains deficient of proteases, especially exo-
proteases, capable of cleaving the modified lipolytic enzyme at a ate close to the peptide addition
(in certain case being the propeptide). Especially relevante host cells are the host cells deficient of
the tripeptidyl-aminopeptidases (TPAP) (see eg. WO 96/14404 from Novo Nordisk A/S),
deficient of dipeptidyl-aminopeptidases (DPAP), or host ceDs deficient of a Kex2 protease or a
Kex2-Gke protease and therefore not capable of cleaving at di-basic site such as Arg-Arg (RR) .

Other examples of host cells include the aspartic proteinase deficient host cells described
in EP 429 490 (from Genencor Inc), host cells deficient of proteolytic enzymes such as the host
cells described in WO 93/00925 (Amgen), WO 92/17595 (Salk Inst Biotech), EP 341 215, EP
574 347, and PCT/DK96/001 1 1 (Novo Nordisk A/S).

In the Examples below an Aspergillus oryzae host cell, with a deleted alkaline protease

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gene, have been used.

Methods of Production

The present invention also relates to methods for producing a modified lipolytic enzyme

5 of the invention comprising (a) cultivating a host cell transformed with a DNA sequence
encoding the enzyme under conditions conducive to expression of the modified lipolytic enzyme;
and (b) recovering the modified lipolytic enzyme.

The host cells may be cultivated in a nutrient medium suitable for production of the
modified lipolytic enzyme using methods known in the art. For example, the cell may be

10 cultivated by shake flask cultivation, small-scale or large-scale fermentation (including
continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors
performed in a suitable medium and under conditions allowing the modified lipolytic enzyme to
be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising
carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e.g.,

is references for bacteria and yeast; Bennett, J.W. and LaSure, L., editors, Mare Gem
Manipulations in Fungi, Academic Press, CA, 1991). Suitable media are available from
commercial suppliers or may be prepared according to published compositions (e.g., in
catalogues of the American Type Culture Collection). If the modified lipolytic enzyme is secreted
into the nutrient medium, the modified lipolytic enzyme can be recovered directly from the

20 medium. If the modified lipolytic enzyme is not secreted, it is recovered from cell lysates.

The resulting modified lipolytic enzyme may be recovered by methods known in the art.
For example, the modified lipolytic enzyme may be recovered from the nutrient medium by
conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-
drying, evaporation, or precipitation The recovered modified lipolytic enzyme may then be

25 further purified by a variety of chromatographic procedures, e.g., ion exchange chromatography,
gel filtration chromatography, affinity chromatography, or the like.

The modified lipolytic enzymes of the present invention may be purified by a variety of
procedures known in the art including, but not limited to, chromatography {e.g y ion exchange,
affinity, hydrophobic, chromatofocuang, and size exclusion), dectrophoretic procedures (e.g,

30 preparative isoelectric focusing (EF), differential solubility (e.g., ammonium sulfate
precipitation), or extraction (see, e.# f Protein Purification, J.-C Janson and Lars Ryden, editors,
VCH Publishers, New York, 1989).

In a preferred embodiment of the invention a peptide addition is applied to the parent enzyme

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by cultivating a host cell comprising a DNA sequence encoding the pre, pro or prepro-form of
the parent lipolytic enzyme, the DNA sequence optionally being present on a vector, and
recovering the resulting modified lipolytic enzyme. The host cell, cultivation condition and/or
recovery conditions being selected so that at the most a partial processing of the pre, pro or
5 preproform of the parent enzyme has occured resulting in that at least 5%, such as at least 10%,
such as at least 15%, such as at least 20%, such as at least 25%, such as at least 50% or at least
75% of the produced modified enzyme molecules comprises the desired , e.g. the entire pre-
sequence or a substantial part thereof.

10 Enzyme composition of the invention

In a further aspect the invention relates to an enzyme composition comprising an enzyme
with lipolytic activity of the invention.

As defined herein, a "substantially pure" enzyme is an enzyme which is essentially free of
other homologous contaminants (originating from the same source as the modified lipolytic

15 enzyme), e.g. , at least about 20% pure, preferably at least about 40% pure, more preferably about
60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even
most preferably about 95% pure, as determined by SDS-PAGE.

In certain cases the host cell does not process all of the modified lipolytic enzyme
molecules expressed by that host at the same cleavage site, resulting in a modified ehzyme

20 product consisting of a portion having the full length peptide addition and one or more other
portions with only a part of the peptide addition. The inventors found that this does not
influence the wash performance significantly. Consequently, even though not all of the
lipolytic enzyme of the enzyme composition of the invention may have retained the full length
peptide addition the enzyme composition is still capable of exerting the desired effect, such as

25 an improved wash performance. Actually, it has been found that as long as at least about 5%
of the total amount of modified lipolytic enzyme of the invention to be used for a given
purpose has the intact peptide addition as disclosed above, this may be found to be sufficient
for providing the desired effect The remaining part of the modified lipolytic enzyme
molecules may then have a peptide addition which is shorter than the one intended (e.g. as a

30 consequence of one or more amino add residues have been cut off during processing of the
enzyme by the host organism) or no peptide addition at all. Therefore, the enzyme
composition of the invention need only to comprise at least about 5%, preferably at least
about 10%, such as at least about 25%, better at least about 50%, especially at least about

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75% of the modified lipolytic enzyme with its full length addition.

Said enzyme composition may further comprise an enzyme selected from the group of
proteases, cellulases, peroxidases, cutinases, amylases and/or lipases, and when intended for
washing also ingredients normally used in detergent compositions.

5 Modified lipolytic enzymes of the invention have been found to be of particular interest as
components in detergent compositions such as washing powder or dishwashing compositions
which will be described in details in the following section. In addition, due to their improved
properties the modified lipolytic enzymes of the invention are contemplated to be useful in,
for example, the baking industry, as a catalyst in organic syntheses (e.g. esterification,

to transesterification or ester hydrolysis reactions), in the papermaking industry (e.g. for pitch
removal), and in the leather, wool and related industries (e.g. for degreasing of animal hides,
sheepskin or wool), and for other applications involving degreasing/defatting.

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DETERGENT DISCLOSURE
Surfactant system

The detergent compositions according to the present invention comprise a surfactant
system wherein the surfactant can be selected from nonionic and/or anionic and/or
cationic and/or ampholytic and/or zwitterionic and/or semi-polar surfactants.

10 The surfactant is typically present at a level of from 0, 1 % to 60% by weight.

The surfactant is preferably formulated to be compatible with enzyme components present
in the composition. In liquid or gel compositions the surfactant is most preferably
formulated such that it promotes, or at least does not degrade, the stability of any enzyme
15 in these compositions.

Preferred surfactant systems to be used according to the present invention comprise as a
surfactant one or more of the nonionic and/or anionic surfactants described herein.

20 Nonionic detergent surfactants normally consist of a water-solubilizing polyalkoxylene or
a mono- or di-alkanolamide group in chemical combination with an organic hydrophobic
group derived, for example, from alkylphenols in which the alkyl group contains from
about 6 to about 12 carbon atoms, dialkylphenols in which each alkyl group contains
from 6 to 12 carbon atoms, primary, sedondary or tertiary aliphatic alcohols (or alkyl-

25 capped derivatives thereof), preferably having from 8 to 20 Carbon atoms,
monocarboxylic acids having from 10 to about 24 carbon atoms in the alkyl group and
polyproxylenes. Also common are fatty acid mono- and dialkanolamides in which the
alkyl group of the fatty acid radical contains from 10 to about 20 carbon atoms and the
alkyloyl group having from 1 to 3 carbon atoms. Optionally, in any of the mono- and di-

30 alkanolamide derivatives there may be a polyoxyalkylene moiety joining the latter groups
and the hydrophobic part of the molecule. In all polyalkoxylene containing surfactants,
the polyalkoxylene moiety preferably consists of from 2 to 20 groups of ethylene oxide or
of ethylene oxide and propylene oxide groups.

35 Polyethylene, polypropylene, and polybutylene oxide condensates of alkyl phenols are
suitable for use as the nonionic surfactant of the surfactant systems of the present inven-
tion, with the polyethylene oxide condensates being preferred. These compounds include

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the condensation products of alkyl phenols having an alkyl group containing from about 6
to about 14 carbon atoms, preferably form about 8 to about 14 carbon atoms, in either a
straightchain or branched-chain configuration with the alkylene oxide. In a preferred
embodiment, the ethylene oxide is present in an amount equal to from about 2 to about
5 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of
alkyl phenol. Commercially available nonionic surfactants of this type include Igepal™
CO-630, marketed by the GAF Corporation; and Triton™ X-45, X-114, X-100 and X-
102, all marketed by the Rohm & Haas Company. These surfactants are commonly
referred to as alkylphenol alkoxylates (e.g., alkyl phenol ethoxylates).

The condensation products of primary and secondary aliphatic alcohols with from about 1
to about 25 moles of ethylene oxide are suitable for use as the nonionic surfactant of the
nonionic surfactant systems of the present invention. The alkyl chain of the aliphatic
alcohol can either be straight or branched, primary or secondary, and generally contains

is from about 8 to about 22 carbon atoms. Preferred are the condensation products of
alcohols having an alkyl group containing from about 8 to about 20 carbon atoms, more
preferably from about 10 to about 18 carbon atoms, with from about 2 to about 10 moles
of ethylene oxide per mole of alcohol. About 2 to about 7 moles of ethylene oxide and
most preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are present in

20 said condensation products. Examples of commercially available nonionic surfactants of
this type include Tergitol™ 15-S-9 (The condensation product of C n -C I5 linear alcohol
with 9 moles ethylene oxide), Tergitol™ 24-L-6 NMW (the condensation product of C 12 -
C M primary alcohol with 6 moles ethylene oxide with a narrow molecular weight
distribution), both marketed by Union Carbide Corporation; Neodol™ 45-9 (the con-

25 densation product of C 14 -C I5 linear alcohol with 9 moles of ethylene oxide), Neodol™ 23-
3 (the condensation product of C 12 -C l3 linear alcohol with 3.0 moles of ethylene oxide),
Neodol™ 45-7 (the condensation product of C 14 -Cu linear alcohol with 7 moles of
ethylene oxide), Neodol™ 45-5 (the condensation product of C, 4 -C,5 linear alcohol with 5
moles of ethylene oxide) marketed by Shell Chemical Company, Kyro™ EOB (the

30 condensation product of C^-C^ alcohol with 9 moles ethylene oxide), marketed by The
Procter & Gamble Company, and Genapol LA 050 (the condensation product of Ci 2 -C, 4
alcohol with 5 moles of ethylene oxide) marketed by Hoechst Preferred range of HLB in
these products is from 8-1 1 and most preferred from 8-10.

35 The detergent composition of the invention may comprise a nonionic material which is
alkoxylated aliphatic alcohols containing at least 25 alkylene oxide groups, preferably at
least 50 alkylene oxide groups, and more preferably at least 80 alkylene oxide groups.

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Also useful as the nonionic surfactant of the surfactant systems of the present invention
are alkylpolysaccharides disclosed in US 4,565,647, having a hydrophobic group
containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16
carbon atoms and a polysaccharide, e.g. a polyglycoside, hydrophiUic group containing
from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from
about 1.3 to about 2.7 saccharide units. Any reducing saccharide containing 5 or 6
carbon atoms can be used, e.g., glucose, galactose and galactosyl moieties can be substi-
tuted for the glucosyl moieties (optionally the hydrophobic group is attached at the 2-, 3-,
4*, etc. positions thus giving a glucose or galactose as opposed to a glucoside or
galactoside). The intersaccharide bonds can be, e.g., between the one position of the
additional saccharide units and the 2-, 3-, 4-, and/or 6- positions on the preceding sac-
charide units.

Another useful nonionic surfactant is a glycoside of a uronic acid, a uronic acid salt or a
uronic acid lactone or polyuronic acid with a straight-chain or branced saturated or unsa-
turated aliphatic chain of from 6 to 24 carbon atoms, optionally containing an aromatic,
cycloaliphatic, mixed aromatic-aliphatic or polyalkyloxyalkyl radical, as described in WO
95/10524.

The preferred alkylpolyglycosides have the formula

R^C^OXCglycosyl),

wherein R 2 is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl,
hydroxyalkylphenyl, and mixtures thereof in which the alkyl groups contain from about
10 to about 18, preferably from about 12 to about 14, carbon atoms; n is 2 or 3,
preferably 2; t is from 0 to about 10, preferably 0; and x is from about 1.3 to about 10,
preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7. The
glycosyl is preferably derived from glucose. To prepare these compounds, the alcohol or
alkylpolyethoxy alcohol is formed first and then reacted with glucose, or a source of
glucose, to form the glucoside (attachment at the 1-position). The additional glycosyl
units can then be attached between their 1-position and the preceding glycosyl units 2-, 3-
, 4-, and/or 6-position, preferably predominately the 2-position.

The condensation products of ethylene oxide with a hydrophobic base formed by the
condensation of propylene oxide with propylene glycol are also suitable for use as the

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additional nonionic surfactant systems of the present invention. The hydrophobic portion
of these compounds will preferably have a molecular weight of from about 1500 to about
1800 and will exhibit water insolubility. The addition of polyoxyethylene moieties to this
hydrophobic portion tends to increase the water solubility of the molecule as a whole, and
the liquid character of the product is retained up to the point where the polyoxyethylene
content is about 50% of the total weight of the condensation product, which corresponds
to condensation with up to about 40 moles of ethylene oxide. Examples of compounds of
this type include certain of the commercially available Pluronic™ surfactants, marketed
by BASF.

Also suitable for use as the nonionic surfactant of the nonionic surfactant system of the
present invention, are the condensation products of ethylene oxide with the product re-
sulting from the reaction of propylene oxide and ethylenediamine. The hydrophobic
moiety of these products consists of the reaction product of ethylenediamine and excess
propylene oxide, and generally has a molecular weight of from about 2500 to about 3000.
This hydrophobic moiety is condensed with ethylene oxide to the extent that the
condensation product contains from about 40% to about 80% by weight of polyoxyet-
hylene and has a molecular weight of from about 5,000 to about 11. (XX). Examples of
this type of nonionic surfactant include certain of the commercially available Tetronic™
compounds, marketed by BASF.

Preferred for use as the nonionic surfactant of the surfactant systems of the present
invention are polyethylene oxide condensates of alkyl phenols, condensation products of
primary and secondary aliphatic alcohols with from about 1 to about 25 moles of
ethyleneoxide, alkylpolysaccharides, and mixtures hereof. Most preferred are C 8 -C, 4
alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C 8 -C, B alcohol
ethoxylates (preferably Ci 0 avg.) having from 2 to 10 ethoxy groups, and mixtures
thereof. Highly preferred nonionic surfactants are polyhydroxy fatty acid amide
surfactants of the formula

R 2 - C - N - Z,

I I
0 R 1

wherein R l is H, or R 1 is C M hydrocarbyl, 2-hydroxy ethyl, 2-hydroxy propyl or a
mixture thereof, R 2 is C5.31 hydrocarbyl, and Z is a polyhydroxyhydrocarbyl having a
linear hydrocarbyl chain with at least 3 hydroxyls directly connected to the chain, or an
alkoxylated derivative thereof. Preferably, R 1 is methyl, R 2 is straight Ci M5 alkyl or C I6 . 1B
alkyl or alkenyl chain such as coconut alkyl or mixtures thereof, and Z is derived from a

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reducing sugar such as glucose, fructose, maltose, lactose, in a reductive amination
reaction.

A nonionic surfactant system comprising an alkoxylated nonionic surfactant having an
average degree of alkoxylation of at least 6 and an aldobionamide of the structure
ANR,R 2 wherein A is a sugar moiety which is an aldobionic acid except that it does not
contain the OH group normally extending from the carbonyl group on the aldonic acid.
NR,R 2 is attached where the hydroxyl group on the aldobionic acid would normally be
found. R,R 2> may be the same or different, is a hydrogen atom, an aliphatic hydrocarbon
radical, an aromatic radical a cycloaliphatic radical an amino acid ester, or an ether
amine. R, and R 2 cannot both be hydrogen atoms as described in WO 95/2770.

Other so-called nonionic detergent compounds include long chain tertiary amine oxides,
long chain tertiary phosphine oxides and dialkyl sulphoxides.

Highly preferred anionic surfactants include alkyl alkoxylated sulfate surfactants hereof
are water soluble salts or adds of the formula ROCA^pjM wherein R is an
unsubstituted C lff C^ alkyl or hydroxyalkyl group having a C^-C^ alkyl component,
preferably a C^-Qo alkyl or hydroxyalkyl, more preferably C ir C, g alkyl or
hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically between
about 0.5 and about 6, more preferably between about 0.5 and about 3, and M is H or a
cation which can be, for example, a metal cation (e.g., sodium, potassium, lithium,
calcium, magnesium, etc.), ammonium or substituted-arnmonium cation. Alkyl ethoxy-
lated sulfates as well as alkyl propoxylated sulfates are contemplated herein. Specific
examples of substituted ammonium cations include methyl-, dimethyl, trimethyl-
ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and
dimethyl piperdinium cations and those derived from alkylamines such as ethylamine,
diethylamine, triethylamine, mixtures thereof, and the like. Exemplary surfactants are
C I2 -C IS alkyl polyethoxylate (1.0) sulfate (C 12 -C 18 E(1.0)M), C l2 -C I8 alkyl polyethoxylate
(2.25) sulfate (C l2 -C IS (2.25)M, and C 12 -C l8 alkyl polyethoxylate (3.0) sulfue (C l2 -
C I8 E(3.0)M), and C i: -C 18 alkyl polyethoxylate (4.0) sulfate (C l2 -C 1S E(4.0)M), wherein M
is conveniently selected from sodium and potassium. Suitable anionic surfactants to be
used are alkyl ester sulfonate surfactants including linear esters of Q-C^ carboxylic acids
(i.e., fatty acids) which are sulfonated with gaseous SO, according to "The Journal of the
American Oil Chemists Society", 52 (1975), pp. 323-329. Suitable starting materials
would include natural fatty substances as derived from tallow, palm oil, etc.

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The preferred alkyl ester sulfonate surfactant, especially for laundry applications,
comprise alkyl ester sulfonate surfactants of the structural formula:

0
I

R 3 - CH - C - OR 4
I

SO3M

wherein R 3 is a Cg-C^ hydrocarbyl, preferably an alkyl, or combination thereof, R 4 is a
C,-C 6 hydrocarbyl, preferably an alkyl, or combination thereof, and M is a cation which
forms a water soluble salt with the alkyl ester sulfonate. Suitable salt-forming cations
include metals such as sodium, potassium, and lithium, and substituted or unsubstituted
ammonium cations, such as monoethanolamine, diethonolamine, and triethanolamine.
Preferably, R 3 is C l0 -C, 6 alkyl, and R 4 is methyl, ethyl or isopropyl. Especially preferred
are the methyl ester sulfonates wherein R 3 is C 10 -C 16 alkyl.

Other suitable anionic surfactants include the alkyl sulfate surfactants which are water
soluble salts or acids of the formula ROS0 3 M wherein R preferably is a C^-C^
hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C t0 -C^ alkyl component, more
preferably a C, r C J8 alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metal
cation (e.g. sodium, potassium, lithium), or ammonium or substituted ammonium (e.g.
methyl-, dimethyl-, and trimethyl ammonium cations and quaternary ammonium cations
such as tetramethyl-ammonium and dimethyl piperdinium cations and quaternary
ammonium cations derived from alkylamines such as ethylamine, diethylamine,
triethylamine, and mixtures thereof, and the like). Typically, alkyl chains of C I2 -C,« are
preferred for lower wash temperatures (e.g. below about 50 o Q and C I6 -C I8 alkyl chains
are preferred for higher wash temperatures (e.g. above about 50°Q.

Other anionic surfactants useful for detersive purposes can also be included in the laundry
detergent compositions of the present invention. Theses can include salts (including, for
example, sodium, potassium, ammonium, and substituted ammonium salts such as mono-
di- and triethanolamine salts) of soap, Cg-C^ primary or secondary alkanesulfonates, CV
C24 olefinsulfonates, sulfonated polycarboxylic acids prepared by sulfonation of the
pyrolyzed product of alkaline earth metal citrates, e.g., as described in GB 1,082,179,
Q-C^ alkylpolyglycolethersulfates (containing up to 10 moles of ethylene oxide); alkyl
glycerol sulfonates, fatty acyl glycerol sulfonates, fatty oleyl glycerol sulfates, alkyl

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phenol ethylene oxide ether sulfates, paraffin sulfonates, alkyl phosphates, isethionates
such as the acyl isethionates, isethionates esters of alkoxy carboxylic acids (as described
in W095/14661), N-acyl taurates, alkyl succinamates and sulfosuccinates, monoesters of
sulfosuccinates (especially saturated and unsaturated C, 2 -C 18 monoesters) and diesters of

5 sulfosuccinates (especially saturated and unsaturated C 6 -C 12 diesters), acyl sarcosinates,
oleoyl sarcosinate, sulfates of alkylpolysaccharides such as the sulfates of
alkylpolyglucoside (the nonionic nonsulfoted compounds being described below),
branched primary alkyl sulfates, and alkyl polyethoxy carboxylates such as those of the
formula RO(CH 2 CH 2 O) k -CH 2 C00-M+ wherein R is a Cg-C^ alkyl, k is an integer from 1

10 to 10, and M is a soluble salt forming cation. Resin acids and hydrogenated resin acids
are also suitable, such as rosin, hydrogenated rosin, and resin acids and hydrogenated
resin acids present in or derived from tall oil. Alkylbenzene sulfonate is highly preferred,
in particular linear alkyl benzene sulfonate (LAS) where the alkyl group contains
preferably from 10 to 18 carbon atoms.

Further examples are described in "Surface Active Agents and Detergents" (Vol. I and II
by Schwartz, Perrry and Berth). A variety of such surfactants are also generally disclosed
in US 3,929,678, (Column 23, line 58 through Column 29, line 23).

20 When included therein, the laundry detergent compositions of the present invention

typically comprise from about 1 % to about 40%, preferably from about 3% to about 20%

by weight of such anionic surfactants.

The laundry detergent compositions of the present invention may also contain cationic,
25 ampholytic, zwitterionic, and semi-polar surfactants, as well as the nonionic and/or
anionic surfactants other than those already described herein.

Cationic detersive surfactants suitable for use in the laundry detergent compositions of the
30 present invention are those having one long-chain hydrocarbyl group. Examples of such
cationic surfactants include the ammonium surfactants such as alkyltrimethylammonium
halogenides, and those surfactants having the formula:

[R 2 (OR 3 ) y ][R 4 (OR 3 ) y l2R 5 N+X-

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wherein R 2 is an alkyl or alkyl benzyl group having from about 8 to about 18 carbon
atoms in the alkyl chain, each R 3 is selected form the group consisting of -CH 2 CH 2 -, -
CH 2 CH(CH 3 h -CH 2 CH(CH 2 OH)-, -CH 2 CH 2 CH 2 -, and mixtures thereof; each R 4 is
selected from the group consisting of C r C 4 alkyl, C r C 4 hydroxyalkyl, benzyl ring
5 structures formed by joining the two R 4 groups, -CH 2 CHOHCHOHCOR 6 CHOHCH 2 OH
wherein R 6 is any hexose or hexose polymer having a molecular weight less than about
1000, and hydrogen when y is not 0; R 5 is the same as R 4 or is an alkyl chain wherein the
total number of carbon atoms or R 2 plus R 5 is not more than about 18; each y is from 0 to
about 10 and the sum of the y values is form 0 to about 15; and X is any compatible
10 anion.

Highly preferred cationic surfactants are the water soluble quaternary ammonium
compounds useful in the present composition having the formula:

* 5 R^Rjig^X* (i)

wherein R t is CVC 16 alkyl, each of R 2 , R 3 and R« is independently C r C 4 alkyl, C r C 4
hydroxy alkyl, benzyl, and -(CyU^H where x has a value from 2 to 5, and X is an
anion. Not more than one of R 2 , R 3 or R< should be benzyl.

The preferred alkyl chain length for R, is C l2 -C 15 particularly where the alkyl group is a
mixture of chain lengths derived from coconut or palm kernel fat or is derived
synthetically by olefin build up or OXO alcohols synthesis.

25 Preferred groups for R^ and are methyl and hydroxyethyl groups and the anion X
may be selected from halide, methosulphate, acetate and phosphate ions. Examples of
suitable quaternary ammonium compounds of formulae (i) for use herein are:

coconut trimethyl ammonium chloride or bromide;

coconut methyl dihydroxyethyl ammonium chloride or bromide;
30 decyl triethyl ammonium chloride;

decyl dimethyl hydroxyethyl ammonium chloride or bromide;

C l2 . 15 dimethyl hydroxyethyl ammonium chloride or bromide;

coconut dimethyl hydroxyethyl ammonium chloride or bromide;

myristyl trimethyl ammonium methyl sulphate;
35 lauryl dimethyl benzyl ammonium chloride or bromide;

lauryl dimethyl (ethenoxy) 4 ammonium chloride or bromide;

choline esters (compounds of formula CO wherein R, is

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CH r CH 2 -0-C-C I2I4 alkyl and R^R* are methyl),
fl

di-alkyl imidazolines [compounds of formula (i)].

Other cationic surfactants useful herein are also described in US 4,228,044 and in EP 000
224.

When included therein, the laundry detergent compositions of the present invention
typically comprise from 0.2% to about 25%, preferably from about 1% to about 8% by
weight of such cationic surfactants.

Ampholyte surfactants are also suitable for use in the laundry detergent compositions of
the present invention. These surfactants can be broadly described as aliphatic derivatives
of secondary or tertiary amines, or aliphatic derivatives of heterocyclic secondary and
tertiary amines in which the aliphatic radical can be straight- or branched-chain. One of
the aliphatic substituents contains at least about 8 carbon atoms, typically from about 8 to
about 18 carbon atoms, and at least one contains an anionic water-solubilizing group, e.g.
carboxy, sulfonate, sulfate. See US 3,929,678 (column 19, lines 18-35) for examples of
ampholytic surfactants.

When included therein, the laundry detergent compositions of the present invention
typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by
weight of such ampholytic surfactants.

Zwitterionic surfactants are also suitable for use in laundry detergent compositions. These
surfactants can be broadly described as derivatives of secondary and tertiary amines, der-
ivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary
ammonium, quaternary phosphonium or tertiary sulfonium compounds. See US
3,929,678 (column 19, line 38 through column 22, line 48) for examples of zwitterionic
surfactants.

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Semi-polar nonionic surfactants are a special category of nonionic surfactants which
include water-soluble amine oxides containing one alkyl moiety of from about 10 to about
18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and
hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; watersoluble
phosphine oxides containing one alkyl moiety of form about 10 to about 18 carbon atoms
and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl
groups containing from about 1 to about 3 carbon atoms; and water-soluble sulfoxides
containing one alkyl moiety of from about 10 to about 18 carbon atoms and a moiety
selected from the group consisting of alkyl and hydroxyalkyl moieties of from about 1 to
about 3 carbon atoms.

Semi-polar nonionic detergent surfactants include the amine oxide surfactants having the
formula:

O
I

R 3 (OR 4 )xN(R 5 )2

wherein R 3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures thereof
containing from about 8 to about 22 carbon atoms; R 4 is an alkylene or hydroxyalkylene
group containing from about 2 to about 3 carbon atoms or mixtures thereof; x is from 0
to about 3: and each R 5 is an alkyl or hydroxyalkyl group containing from about 1 to
about 3 carbon atoms or a polyethylene oxide group containing from about 1 to about 3
ethylene oxide groups. The R 5 groups can be attached to each other, e.g., through an
oxygen or nitrogen atom, to form a ring structure.

These amine oxide surfactants in particular include C I0 -C 18 alkyl dimethyl amine oxides
and C 8 -C, 2 alkoxy ethyl dihydroxy ethyl amine oxides.

Builder system

The compositions according to the present invention may further comprise a builder

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system. Any conventional builder system is suitable for use herein including
aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as
ethylenediamine tetraacetate, metal ion sequestrants such as aminopolyphosphonates,
particularly ethylenediamine tetramethylene phosphonic acid and diethylene triamine
pentamethylenephosphonic acid. Phosphate builders can also be used herein, e.g
pyrophosphates, orthophosphates or polyphosphates.

Suitable builders can be an inorganic ion exchange material, commonly an inorganic
hydrated aluminosilicate material, more particularly a hydrated synthetic zeolite such as
hydrated zeolite A, X, B, HS or MAP. Another suitable inorganic builder material is
layered silicate, e.g. SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consisting of
sodium silicate (Na^Oj).

Suitable polycarboxylates containing one carboxy group include lactic acid, glycolic acid
and ether derivatives thereof as disclosed in BE 831,368, BE 821,369 and BE 821,370.
Polycarboxylates containing two carboxy groups include the water-soluble salts of
succinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid, diglycollic acid,
tartaric acid, tartronic acid and fumaric acid, as well as the ether carboxylates described
in DE 2,446,686, DE 2,446,487, US 3,935,257 and the sulfinyl carboxylates described
in BE 840,623. Polycarboxylates containing three carboxy groups include, in particular,
water-soluble citrates, aconitrates and citraconates as well as succinate derivatives such as
the carboxymethyloxysuccinates described in GB 1,379,241, lactoxysuccinates described
in NL application 7205873, and the oxypolycarboxylate materials such as 2-oxa-l,l,3-
propane tricarboxylates described in GB 1,387,447.

Polycarboxylates containing four carboxy groups include oxydisuccinates disclosed in GB
1,261,829, 1,1,2,2,-ethane tetracarboxylates, 1,1,3,3-propane tetracarboxylates
containing sulfo substituents include the sulfosuccinate derivatives disclosed in GB
1,398,421 and GB 1,398,422 and in US 3,936,448, and the sulfonated pyrolysed citrates
described in GB 1,082,179, while polycarboxylates containing phosphone substituents are
disclosed in GB 1,439,000.

Alicyclic and heterocyclic polycarboxylates include cyclopentane-cis,cis-cis-
tetracarboxylates, cyclopentadienide pentacarboxylates, 2,3,4,5-tetrahydro-furan - cis,
cis, cis-tetracarboxylates, 2,5-tetrahydro-furan-cis, discarboxylates, 2,2,5,5,-
tetrahydrofuran - tetracarboxylates, 1,2,3,4, 5, 6-hexane - hexacarboxylates and car-
boxymethyl derivatives of polyhydric alcohols such as sorbitol, mannitol and xylitol.

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Aromatic polycarboxylates include mellitic acid, pyromellitic acid and the phthalic acid
derivatives disclosed in GB 1,425,343. Of the above, the preferred polycarboxylates are
hydroxycarboxylates containing up to three carboxy groups per molecule, more
particularly citrates.

Preferred builder systems for use in the present compositions include a mixture of a
water-insoluble aluminosilicate builder such as zeolite A or of a layered silicate (SKS-6),
and a water-soluble carboxylate chelating agent such as citric acid.

A suitable chelant for inclusion in the detergent compositions in accordance with the
invention is ethylenediamine-N.N'-disuccinic acid (EDDS) or the alkali metal, alkaline
earth metal, ammonium, or substituted ammonium salts thereof, or mixtures thereof.
Preferred EDDS compounds are the free acid form and the sodium or magnesium salt
thereof. Examples of such preferred sodium salts of EDDS include NajEDDS and
Na4EDDS. Examples of such preferred magnesium salts of EDDS include MgEDDS and
Mg 2 EDDS. The magnesium salts are the most preferred for inclusion in compositions in
accordance with the invention.

Preferred builder systems include a mixture of a water-insoluble aluminosilicate builder
such as zeolite A, and a water soluble carboxylate chelating agent such as citric acid.

Other builder materials that can form part of the builder system for use in granular
compositions include inorganic materials such as alkali metal carbonates, bicarbonates, si-
licates, and organic materials such as the organic phosphonates, amino polyalkylene
phosphonates and amino polycarboxylates.

Other suitable water-soluble organic salts are the homo- or co-polymeric acids or their
salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated
form each other by not more than two carbon atoms.

Polymers of this type are disclosed in GB- A- 1,596,756. Examples of such salts are
polyacrylates of MW 2000-5000 and their copolymers with maleic anhydride, such
copolymers having a molecular weight of from 20,000 to 70,000, especially about
40,000.

Detergency builder salts are normally included in amounts of from 5% to 80% by weight
of the composition. Preferred levels of builder for liquid detergents are from 5% to 30%.

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Enzymes

Preferred detergent compositions, in addition to the enzyme of the invention, comprise
other enzyme(s) which provides cleaning performance and/or fabric care benefits. Such
enzymes include proteases, Upases, cutinases, amylases, cellulases, peroxidases, oxidases
(e.g. laccases).

Proteases : Any protease suitable for use in alkaline solutions can be used. Suitable
proteases include those of animal, vegetable or microbial origin. Microbial origin is pre-
ferred. Chemically or genetically modified mutants are included. The protease may be a
serine protease, preferably an alkaline microbial protease or a trypsin-like protease.
Examples of alkaline proteases are subtilisins, especially those derived from Bacillus .
e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168
(described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of por-
cine or bovine origin) and the Fusarium protease described in WO 89/06270. Preferred
commercially available protease enzymes include those sold under the tradenames
Alcalase, Savinase, Primase, Durazym, and Esperase by Novo Nordisk A/S (Denmark),
those sold under the tradename Maxatase, Maxacal, Maxapem and Properase by Gist-
Brocades/Genencor, those sold under the tradename Purafect and Purafect OXP by
Genencor International, and those sold under the tradename Opticlean and Optimase by
Solvay Enzymes. Protease enzymes may be incorporated into the compositions in
accordance with the invention at a level of from 0.0001% to 2% of enzyme protein by
weight of the composition, in particular at a level of from 0.001% to 0.1% of enzyme
protein by weight of the composition.

Lipases: Any lipase suitable for use in alkaline solutions can be used. Suitable lipases
include those of bacterial or fungal origin and chemically or genetically modified lipase
mutants.

Examples of useful lipases include a Humicola lanuginosa lipase, e.g., as described in EP
258 068 and EP 305 216, and mutants thereof as described in WO 92/05249, WO
94/25577 and WO 95/22615 a Rhizomucor miehei lipase, e.g., as described in EP 238
023, a Candida lipase, such as a C. antarctica lipase, e.g., the C. antarctica lipase A or B
described in EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and R
pseudoalcalipenes lipase, e.g., as described in EP 218 272, or any mutant of said
Pseudomonas lipases, a P. cepacia lipase, e.g., as described in EP 331 376, a P. stutzeri

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lipase, e.g., as disclosed in BP 1,372,034, a P. fluorescens lipase, a Bacillus lipase , e.g.,
a B, subtilis lipase (Dartois et al., (1993), Biochemica et Biophysica acta 1131, 253-260),
a B. stearothermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422).
Furthermore, a number of cloned lipases may be useful, including the Penicillium
camembertii lipase described by Yamaguchi et al., (1991), Gene 103, 61-67), the
Geotricum candidum lipase (Schimada, Y. et al., (1989), J. Biochem., 106, 383-388),
and various Rhizopus lipases such as a R. delemar lipase (Hass, M J et al., (1991), Gene
109, 117-113), a R. niveus lipase (Kugimiya et ah, (1992), Biosci. Biotech. Biochem.
56, 716-719) and a R. oryzae lipase.

Other types of lipolytic enzymes such as cutinases may also be useful, e.g., a cutinase
derived from Pseudomonas mendocina as described in WO 88/09367, or a cutinase
derived from Fusarium solani pisi (e.g. described in WO 90/09446). Especially suitable
lipases are lipases such as Ml Lipase™, Luma fast™ and Lipomax™ (Gist-Broca-
des/Genencor), Lipolase™ and Lipolase Ultra™ (Novo Nordisk A/S), and lipase P
"Amano" (Amano Pharmaceutical Co. Ltd.).

The lipases are normally incorporated in the detergent composition at a level of from
0.0001% to 2% of enzyme protein by weight of the detergent composition, in particular
at a level of from 0.001 % to 0. 1 % of enzyme protein by weight of the composition.

Amylases: Any amylase (a and/or b) suitable for use in alkaline solutions can be used.
Suitable amylases include those of bacterial or fungal origin. Chemically or genetically
modified mutants are included. Amylases include, for example, a-amylases obtained from
a special strain of B. licheniformis . described in more detail in GB 1,296,839.
Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ and BAN™
(available from Novo Nordisk A/S) and Rapidase™ and Maxamyl P™ (available from
(Gist-Brocades/Genencor).

The amylases are normally incorporated in the detergent composition at a level of from
0.0001% to 2% of enzyme protein by weight of the detergent composition, in particular
at a level of from 0.001 % to 0. 1 % of enzyme protein by weight of the composition.

Cellulases : Any cellulase suitable for use in alkaline solutions can be used. Suitable
cellulases include those of bacterial or fungal origin. Chemically or genetically modified
mutants are included. Suitable cellulases are disclosed in US 4,435,307, which discloses
fungal cellulases produced from Humicola insolens . Especially suitable cellulases are the

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ceUuIases having color care benefits. Examples of such cellulases are cellulases described
in published European patent application No. 0495257. Commercially available cellulases
is Celluzyme^produced by a strain of Humicola insolent , (Novo Nordisk A/S), and
KAC-SOOOB^CCao Corporation).

Said cellulases are normally incorporated in the detergent composition at a level of from
0.0001% to 2% of enzyme protein by weight of the detergent composition, in particular
at a level of from 0.001 % to 0. 1 % of enzyme protein by weight of the composition.

Peroxidases/Oxidases- Peroxidase and/or oxidase enzymes are used in combination with
hydrogen peroxide or oxygen sources, e.g., percarbonate, perborate, persulfete,
hydrogen peroxide, oxygen, etc. They are used for "Solution bleaching", i.e. to prevent
transfer of a textile dye from a dyed fabric to another fabric when said fabrics are washed
together in a wash liquor, preferably together with an enhancing agent as described in
e.g. WO 94/12621 and WO 95/01426. Suitable peroxidases/oxidases include those of
plant, bacterial or fungal origin. Chemically or genetically modified mutants are inclu-
ded.

Said peroxidase and/or oxidase enzymes are normally incorporated in the detergent
composition at a level of from 0.0001% to 2% of enzyme protein by weight of the
detergent composition, in particular at a level of from 0.001 % to 0. 1 % of enzyme protein
by weight of the composition.

Mixtures of the above mentioned enzymes are encompassed herein, in particular a mix-
ture of a protease, an amylase, a lipase and/or a cellulase.

Optional determent ingredients

Bleachinp aoentr Additional optional detergent ingredients that can be included in the
detergent compositions of the present invention include bleaching agents such as PB1,
PB4 and percarbonate with a particle size of 400-800 microns. These bleaching agent
components can include one or more oxygen bleaching agents and, depending upon the
bleaching agent chosen, one or more bleach activators. When present oxygen bleaching
compounds will typically be present at levels of from about 1 % to about 25%.

The bleaching agent component for use herein can be any of the bleaching agents useful
for detergent compositions including oxygen bleaches as well as others known in the art.

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The bleaching agent suitable for the present invention can be an activated or non-activated
bleaching agent.

One category of oxygen bleaching agent that can be used encompasses percarboxylic acid
bleaching agents and salts thereof. Suitable examples of this class of agents include
magnesium monoperoxyphthalate hexahydrate, the magnesium salt of meta-chloro
perbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid and diperoxydodecanedioic acid.
Such bleaching agents are disclosed in US 4,483,781, US 740,446, EP 0 133 354 and US
4,412,934. Highly preferred bleaching agents also include 6-nonylamino-6-
oxoperoxycaproic acid as described in US 4,634,551.

Another category of bleaching agents that can be used encompasses the halogen bleaching
agents. Examples of hypohalite bleaching agents, for example, include trichloro
isocyanuric acid and the sodium and potassium dichloroisocyanurates and N-chloro and
N-bromo alkane sulphonamides. Such materials are normally added at 0.5-10% by weight
of the finished product, preferably 1-5% by weight.

The hydrogen peroxide releasing agents can be used in combination with bleach activators
such as tetraacetylethylenediamine (TAED), nonanoyloxybenzenesulfonate (NOBS,
described in US 4,412,934), 3,5-trimethylhexsanoloxybenzenesulfonate (ISONOBS,
described in EP 120 591) or pentaacetylglucose (PAG), which are perhydrolyzed to form
a peracid as the active bleaching species, leading to improved bleaching effect. In addi-
tion, very suitable are the bleach activators C8(6-octanamido-caproyl)
oxybenzenesulfonate, C9(6-nonanamido caproyl) oxybenzenesulfonate and C10 (6-
decanamido caproyl) oxybenzenesulfonate or mixtures thereof. Also suitable activators
are acylated citrate esters such as disclosed in European Patent Application No.
91870207.7.

Useful bleaching agents, including peroxyacids and bleaching systems comprising bleach
activators and peroxygen bleaching compounds for use in cleaning compositions
according to the invention are described in application USSN 08/136,626.

Imine quaternary salts may used as a bleach catalyst together with a peroxygen compound
as described in WO 95/13352.

The hydrogen peroxide may also be present by adding an enzymatic system (i.e. an

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enzyme and a substrate therefore) which is capable of generation hydrogen peroxide at
the beginning or during the washing and/or rinsing process. Such enzymatic systems are
disclosed in published EP Patent Application No 0537381.

The release of peracid from a peroxygen bleach source may be activated by use of a
lipase of the invention. The necessary components for the enzymatic hydrolysis system is
the peracid precursor substrate: a diacyl peroxide R r CO-OOCO-R, where R and Rj
may be saturated or unsaturated alkyl, an aryl group or an alkaryl. The lipase reacts with
the substrate and releases peracids in the wash.

A non-aqeous liquid detergent composition may comprise a peroxyacid material e.g
peroxyacids consisting of N,N'-Di(4-Percarboxybenzoyl)ethylenediamine (PCBED),
N^'-Terephthaloyl-diCfr-aminopercarboxycaproic acid) (TPCAP), N,N'-Di(4-percar-
boxybenzoyl)piperazine (PCBPIP), N,N'-Di(4-Percarboxybenzoyl)-l ,4-diamino-
cyclohexane (PCBHEX), N,N , -Di(4-Percarboxyboizoyl)-l,4.butanediamine (PCBBD),
N,N'-Di(4-Percarboxyaniline)-terephtalate (DPCAT), N^N'NM^AS-tetra-carboxy-
benzoyl-di(6-aminopercarboxycaproic acid) (DiPAP), N,N'-Di(percarboxy-
adipoyl)phenylenediamine (DPAPD), N,N , -Sucrinoyl-di(4-percarboxy)aniline (SDPCA),
C3 analogue of N,N'-Terephaloyl-di-(8-amino peroxyoctanoic acid) (TPOCT) as
described in WO 95/06104.

Bleaching agents other than oxygen bleaching agents are also known in the art and can be
utilized herein. One type of non-oxygen bleaching agent of particular interest includes
photoactivated bleaching agents such as the sulfonated zinc and/or aluminum
phthalocyanines. These materials can be deposited upon the substrate during the washing
process. Upon irradiation with light, in the presence of oxygen, such as by hanging
clothes out to dry in the daylight, the sulfonated zinc phthalocynaine is activated and,
consequently, the substrate is bleached. Preferred zinc phthalocyanine and a pho-
toactivated bleaching process are described in US 4,033,718. Typically, detergent
composition will contain about 0.025% to about 1.25%, by weight, of sulfonated zinc
phthalocyanine.

Bleaching agents may also comprise a manganese catalyst. The manganese catalyst may,
e.g., be one of the compounds described in "Efficient manganese catalysts for low-
temperature bleaching", Nature 369 . 1994, pp. 637-639.

Suds suppressors: Another optional ingredient is a suds suppressor, exemplified by
silicones, and silica-silicone mixtures. Silicones can be generally represented by alkylated

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polysiloxane materials while silica is normally used in finely divided forms exemplified
by silica aerogels and xerogels and hydrophobic silicas of various types. Theses materials
can be incorporated as particulates in which the suds suppressor is advantageously
releasably incorporated in a water-soluble or waterdispersible, substantially non surface-
active detergent impermeable carrier. Alternatively the suds suppressor can be dissolved
or dispersed in a liquid carrier and applied by spraying on to one or more of the other
components.

A preferred silicone suds controlling agent is disclosed in US 3,933,672. Other
particularly useful suds suppressors are the self-emulsifying silicone suds suppressors,
described in DE 2,646,126. An example of such a compound is DC-544, commercially
available form Dow Corning, which is a siloxane-glycol copolymer. Especially preferred
suds controlling agent are the suds suppressor system comprising a mixture of silicone
oils and 2-alkyl-alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanol which are
commercially available under the trade name Isofol 12 R.

Such suds suppressor system are described in published European Patent Application No.
0593841.

Especially preferred silicone suds controlling agents are described in published European
Patent Application No. 0S73699. Said compositions can comprise a silicone/ silica
mixture in combination with fumed nonporous silica such as Aerosil R .

The suds suppressors described above are normally employed at levels of from 0.001% to
2% by weight of the composition, preferably from 0.01 % to 1 % by weight.

Other components: Other components used in detergent compositions may be employed
such as soil-suspending agents, soil-releasing agents, optical brighteners, abrasives,
bactericides, tarnish inhibitors, coloring agents, and/or encapsulated or nonencapsulated
perfumes.

Especially suitable encapsulating materials are water soluble capsules which consist of a
matrix of polysaccharide and polyhydroxy compounds such as described in GB
1,464,616.

Other suitable water soluble encapsulating materials comprise dextrins derived from
ungelatinized starch acid esters of substituted dicarboxylic acids such as described in US

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3,455,838. These acid-ester dextrins are, preferably, prepared from such starches as
waxy maize, waxy sorghum, sago, tapioca and potato. Suitable examples of said
encapsulation materials include N-Lok manufactured by National Starch. The N-Lok
encapsulating material consists of a modified maize starch and glucose. The starch is
modified by adding monofunctional substituted groups such as octenyl succinic acid
anhydride.

Antiredeposition and soil suspension agents suitable herein include cellulose derivatives
such as methylcellulose, carboxymethylceUulose and hydroxyethylcellulose, and homo- or
co-polymeric polycarboxylic acids or their salts. Polymers of this type include the
polyacrylates and maleic anhydride-acrylic acid copolymers, e.g Sokalan CPS, previously
mentioned as builders, as well as copolymers of maleic anhydride with ethylene,
methylvinyl ether or methacrylic acid, the maleic anhydride constituting at least 20 mole
percent of the copolymer. These materials are normally used at levels of from 0.5% to
10% by weight, more preferably form 0.75% to 8%, most preferably from 1% to 6% by
weight of the composition.

Preferred optical brighteners are anionic in character, examples of which are disodium
4,4 , -bis-(2-diethanolamino-4-anilino -s- triazin-^ylantimOstilbene^' disulphonate, dis-
odium 4, - 4 ' -bis-(2-morpholino^-anilino-s-triazin-6-ylamino-stilbene-2 : 2 * - disul-
phonate, disodium 4,4' - bis-(2,4-<lianilino-s-triazin-6-yIamino)stilbene-2:2 , - disul-
phonate, monosodium 4\4" - bis-(2,4-dianilino-s-tri-azin-6 ylamino)stilbene-2-
sulphonate, disodium 4,4' -bis-(2-anUino-4^(N-methyl-N-2-hydroxyethylamino)-s-triazin-
6-ylamino)stilbene-2,2 , - disulphonate, di-sodium 4,4* -bis-(4-phenyl-2,l,3-triazol-2-yl)-
stiIbene-2,2' disulphonate, di-so-dium 4,4 , bis(2-anilino-4-(l-methyl-2-hydroxy-
ethylanuno)-s-triazin-6-ylami-no)stilbene-2,2 , disulphonate, sodium 2(stilbyl-4"-(naphtho-
1\2':4,5)-1,2,3, - triazole-2"-sulphonateand 4,4'-bis(2-sulphostyryl)biphenyL

Other useful polymeric materials are the polyethylene glycols, particularly those of
molecular weight 1000-10000, more particularly 2000 to 8000 and most preferably about
4000. These are used at levels of from 0.20% to 5% more preferably from 0.25% to
2.5% by weight. These polymers and the previously mentioned homo- or co-polymeric
polycarboxylate salts are valuable for improving whiteness maintenance, fabric ash
deposition, and cleaning performance on clay, proteinaceous and oxidizable soils in the
presence of transition metal impurities.

A graft polymer as described in WO 95/22593 may also be used.

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Soil release agents useful in compositions of the present invention are conventionally
copolymers or terpolymers of terephthalic acid with ethylene glycol and/or propylene gly-
col units in various arrangements. Examples of such polymers are disclosed in US
5 4,116,885, US 4,711,730 and EP 0 272 033. A particular preferred polymer in
accordance with EP 0 272 033 has the formula:

(CH 3 (PEG) 43 ) 0 . 75 (P^^

10 where PEG is -(OQH^O-, PO is (OQI^O) and T is (pcOC^CO).

Also very useful are modified polyesters as random copolymers of dimethyl terephthalate,
dimethyl sulfoisophthalate, ethylene glycol and 1-2 propane diol, the end groups
consisting primarily of sulphobenzoaie and secondarily of mono esters of ethylene glycol
15 and/or propane-diol. The target is to obtain a polymer capped at both end by
sulphobenzoate groups, "primarily in the present context most of said copolymers her-
ein wfll be endcapped by sulphobenzoate groups. However, some copolymers will be less
than fully capped, and therefore their end groups may consist of monoester of ethylene
glycol and/or propane 1-2 diol, thereof consist "secondarily* of such species.

The selected polyesters herein contain about 46% by weight of dimethyl terephthalic acid,
about 16% by weight of propane -1.2 diol, about 10% by weight ethylene gluycol about
13% by weight of dimethyl sulfobenzoic acid and about 15% by weight of
sulfoisophthalic acid, and have a molecular weight of about 3.000. The polyesters and
25 their method of preparation are described in detail in EP 31 1 342.

Softening agents: Fabric softening agents can also be incorporated into laundry detergent
compositions in accordance with the present invention. These agents may be inorganic or
organic in type. Inorganic softening agents are exemplified by the smectite clays

30 disclosed in GB-A-1 400898 and in US 5,019,292. Organic fabric softening agents
include the water insoluble tertiary amines as disclosed in GB-A1 514 276 and EP 0 011
340 and their combination with mono C, 2 -C u quaternary ammonium salts are disclosed in
EP 026 528 and di-long-chain amides as disclosed in EP 0 242 919. Other useful organic
ingredients of fabric softening systems include high molecular weight polyethylene oxide

35 materials as disclosed in EP 0 299 575 and 0 3 13 146.

Levels of smectite clay are normally in the range form 5% to 15%, more preferably form

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8% to 12% by weight, with the material being added as a dry mixed component to the
remainder of the formulation. Organic fabric softening agents such as the water-insoluble
tertiary amines or dilong chain amide materials are incorporated at levels of from 0.5% to
5% by weight, normally from 1% to 3% by weight whilst the high molecular weight
polyethylene oxide materials and the water soluble cationic materials are added at levels
of from 0.1% to 2%, normally from 0.15% to 1.5% by weight. These materials are
normally added to the spray dried portion of the composition, although in some instances
it may be more convenient to add them as a dry mixed particulate, or spray them as
molten liquid on to other solid components of the composition.

Polymeric dye transfer inhibiting apents: The detergent compositions according to the
present invention may also comprise from 0.001% to 10%, preferably from 0.01% to
2%, more preferably form 0.05% to 1% by weight of polymeric dye transfer inhibiting
agents. Said polymeric dye transfer inhibiting agents are normally incorporated into
detergent compositions in order to inhibit the transfer of dyes from colored fabrics onto
fabrics washed therewith. These polymers have the ability to complex or adsorb the
fugitive dyes washed out of dyed fabrics before the dyes have the opportunity to become
attached to other articles in the wash.

Especially suitable polymeric dye transfer inhibiting agents are polyamine N-oxide
polymers, copolymers of N-vinyipyrrolidone and N-vinylimidazole, polyvinylpyrrolidone
polymers, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. Addition
of such polymers also enhances the performance of the enzymes according the invention.

The detergent composition according to the invention can be in liquid, paste, gels, bars or
granular forms. Non-dusting granulates may be produced, e.g., as disclosed in US
4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by
methods known in the art. Examples of waxy coating materials are poly(ethylene oxide)
products (polyethyleneglycol, PEG) with mean molecular weights of 1000 to 20000;
ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty
alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are
15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and
triglycerides of fatty acids. Examples of film-forming coating materials suitable for
application by fluid bed techniques are given in GB 1483591.

Granular compositions according to the present invention can also be in "compact form",
i.e. they may have a relatively higher density than conventional granular detergents, i.e.

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form 550 to 950 g/1; in such case, the granular detergent compositions according to the
present invention will contain a lower amount of "Inorganic filler salt", compared to con-
ventional granular detergents; typical filler salts are alkaline earth metal salts of sulphates
and chlorides, typically sodium sulphate; "Compact" detergent typically comprise not
more than 10% filler salt. The liquid compositions according to the present invention can
also be in "concentrated form", in such case, the liquid detergent compositions according
to the present invention will contain a lower amount of water, compared to conventional
liquid detergents. Typically, the water content of the concentrated liquid detergent is less
than 30%, more preferably less than 20%, most preferably less than 10% by weight of
the detergent compositions.

The compositions of the invention may for example, be formulated as hand and machine
laundry detergent compositions including laundry additive compositions and compositions
suitable for use in the pretreatment of stained fabrics, rinse added fabric softener
compositions, and compositions for use in general household hard surface cleaning
operations and dishwashing operations.

Particular forms of laundry detergent compositions within the invention include:

1) A detergent composition formulated as a granulate having a bulk density of at least
600 g/1 comprising

Linear alkylbenzenesulfonate (calculated as acid)

7 - 12%

Alcohol ethoxysulfate (e.g. C 12 .„

alcohol, 1-2 EO) or alkyl sulfate (e.g. C )6 .,»)

1 - 4%

Alcohol ethoxylate (e.g. C I4 ., 5 alcohol,
7EO)

5 - 9%

| Sodium carbonate (as Na^CO,)

14 - 20%

| Soluble silicate (as N^O^iO^

2 - 6%

Zeolite (as NaAlSiOJ

15 - 22%

Sodium sulfate (as Na^SOJ

0-6%

Sodium citrate/citric acid
(as C 6 H s Na,0 7 /C 6 H,0 7 )

0 - 15%

Sodium perborate (as NaBO,.H 2 0)

11 - i8%

TAED

2 - 6%

Carboxymethylcellulose

Polymers (e»g. maleic/acrylic acid copolymer, PVP,

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

PEG)

0 - 3%

Enzymes (calculated as pure enzyme
protein)

0.0001 -0.1%

Minor ingredients (e.g. suds suppressors, perfume,
optical brightener, photobleach)

0 - 5%

2) A detergent composition formulated as a granulate having a bulk density ol
600 g/1 comprising

Linear aucyiDenzenesulionate (calculated as acid)

6-11%

Alcohol ethoxysulfate (e.g. C n . u
alcohol, 1-2 EO or alkyl sulfate (e.g. C 16 .„)

1- 3%

Alcohol ethoxylate (e.g. C I4 .,j alcohol,
7EO)

5-9%

Sodium carbonate (as NajCO,)

15-21% 1

Soluble silicate (as NajO^iOj)

1-4%

Zeolite (as NaAlSiOJ

24 - 34%

Sodium sulfate (as NajSQ,)

4-10%

Sodium citrate/citric acid
(as QHjNajCVQH.Q,)

0-15%

Carboxymethylcellulose

0- 2%

Polymers (e.g. maleic/acrylic acid copolymer,
PVP, PEG)

1 - 6%

Enzymes (calculated as pure enzyme
protein)

0.0001- 0.1%

Minor ingredients (e.g. suds suppressors, perfume)

0- 5%

3) A detergent composition formulated as a granulate having a bulk density of at least
600 g/1 comprising

Linear alkylbenzenesulfonate (calculated as acid)

5-9% |

Alcohol ethoxylate (e.g. C n . u alcohol,
7EO)

7-14% [

Soap as fatty acid (e.g. C IMI fatty acid)

1 - 3%

Sodium carbonate (as Na 2 CO,)

10 - 17%

| Soluble silicate (as NajO^SiOj)

3 - 9%

| Zeolite (as NaAlSiO*)

23 - 33%

1 Sodium sulfate (as NajS04)

0 -4% |

SUBSTITUTE SHEET (RULE 26)

WO 97/04079

PCT/DK96/00322

oOQiufTi perDoraie ^as iNax>u 3 . ri 2 <Jj

8 - 16%

TAED

2 - 8%

Phosphonate (e.g. EDTMPA)

0 - 1%

Caiboxymethylcellulose

0 - 2%

Polymers (e.g. maleic/acrylic acid copolymer,
| PVP, PEG)

0 - 3%

Enzymes (calculated as pure enzyme
protein)

0.0001 - 0.1%

Minor ingredients (e.g. suds suppressors, perfume,
optical brightener)

0 - 5%

4) A detergent composition formulated as a granulate having a bulk density of
600 g/1 comprising

Linear alkylbenzenesulfonate (calculated as acid)

8 -12%

Alcohol ethoxylate (e.g. C l2 . is alcohol,
7EO)

10 -25%

Sodium carbonate (as Na^Oj)

14 -22%

Soluble silicate (as HaJOJSiOJ

1 - 5%

Zeolite (as NaAlSi0 4 )

25 -35%

Sodium sulfate (as NajSOJ

0 -10%

Carboxymethylcellulose

0 - 2%

Polymers (e.g. maleic/acrylic acid copolymer,
PVP, PEG)

1 - 3%

Enzymes (calculated as pure enzyme
protein)

0.0001- 0.1%

Minor ingredients (e.g. suds suppressors, perfume)

0 - 5%

5) An aqueous liquid detergent composition comprising

Linear alkylbenzenesulfonate (calculated as acid)

15 -21%

Alcohol ethoxylate (e.g. C 12 .u alcohol,
7 EO or C 12l5 alcohol, 5 EO)

12 -18%

| Soap as fatty acid (e.g. oleic acid)

3 - 13%

| Alkenylsuccinic acid (C l2 . l4 )

0 - 13%

| Aminoethanol

8 -18%

| Citric acid

2 - 8%

SUBSTITUTE SHEET (RULE 26)

WO 97/04079

PCT/DK96/00322

Phosphorate

0 - 3%

Polvmer* (e p PVP PPrft

U - 3%

Borate (as B 4 Q,)

0 - 2%

Ethanol

0 - 3%

Propylene glycol

8 - 14%

Enzymes (calculated as pure enzyme
protein)

0.0001 - 0.1%

Minor ingredients (e.g. dispersants, suds
[ suppressors, perfume, optical brightener)

0 - 5% •

6) An aqueous structured liquid detergent composition comprising

Linear alkylbenzenesulfonate (calculated as acid)

15 -21%

Alcohol ethoxylate (e.g. C I2 . 15 alcohol,
7 EO, or C 12 . I5 alcohol, 5 EO)

3 -9*

Soap as fatty acid (e.g. oleic acid)

3 -10%

Zeolite (as NaAlSi0 4 )

14 -22%

Potassium citrate

9 - 18%

Borate (as B 4 0 7 )

0 - 2%

Carboxymethylcellulose

0 - 2%

Polymers (e.g. PEG, PVP)

0 - 3%

Anchoring polymers such as, e.g., lauryl
methacrylate/acrylic acid copolymer; molar ratio
25:1; MW 3800

0 - 3%

Glycerol

0 - 5%

Enzymes (calculated as pure enzyme protein)

0.0001 - 0.1%

Minor ingredients (e.g. dispersants, suds
suppressors, perfume, optical brighteners)

0-3% 1

7) A detergent composition formulated as a granulate having a bulk density of at Is
g/1 comprising

Fatty alcohol sulfate

5-10% B

Ethoxylated fatty acid monoethanolamide

3-9% I

Soap as fatty acid

0-3% I

Sodium carbonate (as Na 2 C0 3 )

5-10% I

| Soluble silicate (as Na.O^SiO^

1-4% |

SUBSTITUTE SHEET (RULE 26)

WO 97/04079

PCT/DK96/00322

Zeolite fas NaAlSifV*

on . ac\<&

XU - H\J70

Sodium sulfate (as NajSOJ

2 - 8%

Sodium perborate (as NaBC^.HjO)

12 - 18%

TAED

2 - 7%

Polymers (e.g. maleic/acrylic acid copolymer,
PEG)

— .

1-5%

Enzymes (calculated as pure enzyme
protein)

0.0001 - 0.1%

Minor ingredients (e.g. optical brightener, suds
suppressors, perfume)

0 - 5%

8) A detergent composition formulated as a granulate comprising

Linear alkylbenzenesulfonate (calculated as acid)

8 • 14%

Ethoxylated fatty acid monoethanolamide

5 -11%

Soap as fatty acid

0-3% 1

Sodium carbonate (as Na 2 CO,)

4-10% |

Soluble silicate (as NajO^SiOj)

1 - 4%

Zeolite (as NaAlSiOJ

30 - 50%

Sodium sulfate (as NajSOJ

3 - 11%

Sodium citrate (as QHjNajOj)

5 - 12%

Polymers (e.g. PVP, maleic/acrylic acid
copolymer, PEG)

1 - 5%

Enzymes (calculated as pure enzyme
protein)

0.0001 - 0.1% 1

Minor ingredients (e.g. suds suppressors, perfume)

0-5% |

9) A detergent composition formulated as granulate

Alkyl sulfate (e.g C 12 . l& )

6 - 12 % 1

Soap, Na-salt

0-3 % |

Nonionic, (eg. Alcohol ethoxylate C 10 ., 8 , 2-7 EO)

2-8 %

Alkylglucamid (e.g C lM8 )

2-6%

Zeolite (NaAlSi0 4 )

14 - 24 %

Sodium carbonate (Na^Q,)

7 - 13 % I

Sodium disillicate; (NajO^iOj)
(e.g SKS6)

10 - 14 % j

SUBSTITUTE SHEET (RULE 26)

WO 97/04079

PCT/DK96/00322

Sodium sulfate (NajSOJ

5-9 %

Sodium percarbonate

10-16%

TAED

1-5%

CMC

0-3 %

Pnl vf*^ rtvi y via t*»

1 T of

1 - 7 %

Polvmers fe e PVP PEG maleie/arrvlir nriH

copolymer)

U " L 70

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001-0.1 %

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

Bulk density (g/1) at least

700

10) A detergent composition formulated as granulate

Alkyl sulfate (e.g C l2 . lg )

7-11% |

Soap, Na-salt

0-3 % 1

Nonionic, (eg. alcohol ethoxylate C 1(M8 , 2-7 EO)

7-11% 1

Alkylglucamid (e.g C 1<tlR )

2-6%

Zeolite (NaAlSiOJ

19 - 29 %

Sodium carbonate (NajCO,)

12 - 18 %

Sodium disillicate; (NasO^SiOj)
(e.g SKS6)

7- 11 %

Sodium sulfate (NajSOJ

5-9 %

CMC

0-3 %

Polycarboxylate

4-8%

Polymers (e.g PVP, PEG, maleic/acrylic acid
J copolymer)

1 -3 %

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001 -0.1 %

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

| Bulk density (g/1) at least

700

SUBSTITUTE SHEET

WO 97/04079 PCT/DK96/00322

11) A detergent composition formulated as granulate

1 - -

Alkyl sulfate (e.g C I2 . ia )

4 - 10 %

Nonionic, (eg. alcohol ethoxylate C I(M g, 2-7 EO)

2-7 %

Phosphate (as STPP)

17-27%

sodium carbonate (Na^Cy

A O Of

4 - 8 %

Sodium disillicate; (Na 2 0:2Si02)
(e.gSKS6)

4-8 %

Sodium sulfate (NajSOJ

1 O *\ S trt

18 - 26 %

Sodium perborate tetrahydrate

13 - 19 %

TAED

1-4 % 1

CMC

0-2 % 1

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001-0.1 % 1

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

Bulk density (g/1) at least

600

12) A detergent composition formulated as granulate

Alkyl sulfuric acid (e.g C ms )

2-9 %

Soap, Na-salt

1 -4 %

Nonionic, (eg. alcohol ethoxylate C l(M8 , 2-7 EO)

9-15 %

Zeolite (NaAlSi0 4 )

35 - 45 %

Sodium carbonate (Na^O,)

3- 10 %

Sodium disillicate; (N^O^iQJ '
(e.g SKS6)

0-4%

Sodium sulfate (Na^O^)

2-6%

Sodium percarbonate

14 - 20 %

TAED

2-7%

I CMC

0-3 %

[j Polycarboxylate

0-2 %

9 Enzymes including modified lipolytic enzymes
1 (calculated as pure enzyme protein)

0.0001 -0.1

I Minor ingrediens (e.g suds supressors, perfume,
j} optical brightener, photobleach)

0-5 % j

SUBSTITUTE SHEF*

WO 97/04079

PCT/DK96/00322

Bulk density (g/1) at least

700

13)

A detergent composition formulated as granulate

Alkyl benzene sulfonic acid

8- 14 %

Alkyl sulfuric acid (e.gC 12 . ia )

2-6%

Nonionic, (eg. alcohol ethoxylate C I(M8 , 2-7 EO)

9- 13 %

Zeolite (NaAlSiOJ (e.g Zeolite 4A)

20 - 30 %

Sodium carbonate (Na^COj)

0-6%

Sodium disillicate; (NajO^SiO,)

0-3 %

Sodium sulfite (NajSO^

0-6%

Sodium perborate tetrahydrate

22 - 28 %

TAED

5-9 %

CMC

0-2 %

Polymers (e.g maleic/acrylic acid copolymer)

0-4 %

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001 -.0.1

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5%

Bulk density (g/1) at least

600

14) A detergent composition formulated as granulate

Alkyl benzene sulfonic acid

6-12%

Alkyl ether sulfuric acid (e.g. C, 2 .„ alcohol, 4-10
EO) or alkyl sulfate (e.g C„. 1S )

2-6%

Soap, Na-salt

0-2 %

Nonionic, (eg. alcohol ethoxylate C I2 .„, 3-10 EO)

9- 13 %

Zeolite (NaAlSi0 4 ) (e.g Zeolite 4A)

39 - 49 %

Sodium carbonate (Na^CO,)

2-8 %

Sodium disillicate; (Na^-.^iOj)

0-3 %

| Sodium sulfate (NajS0 4 )

2-8% I

CMC

0-3% j

Polymers (e.g maleic/acrylic acid copolymer)

0-4%

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001-0.1

| Minor ingrediens (e.g suds supressors, perfume,

0-5 %

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

optical brightener, photobleach)

Bulk density (g/1) at least

600

15) A detergent composition formulated as granulate

Aijcyi suiiate ^e.g c^igj

*\ o fit

2- 8 %

ooap, iNa-sait

0-3 %

Nonionic, (eg. alcohol ethoxylate C, 2 . l6 , 3-10 EO)

10 - 16 %

Zeolite (NaAlSi0 4 ) (e.g Zeolite 4A)

47 - 57 %

Sodium carbonate fNa^PO^

ij - 70

Sodium citrate /citric acid

ft <tL
U - JO 70

Sodium sulfate (Na^OJ

1-5 %

CMC

0-3%

Polycarboxylate

0-2 %

Polymers (e.g PVP)

0-2 %

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001 - 0.1 I

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 % I

Bulk density (g/1) at least

800 g

16) A detergent composition formulated as granulate

Alkyl benzene sulfonic arid

0-3 % 1

Nonionic, (eg. alcohol ethoxylate C,j. l8 , 3-10 EO)

1-5%

Phosphate (as STPP)

12 - 18 %

Sodium carbonate (NajCO,)

16-24% I

Sodium disillicate; (NafizTSiOJ

1-3% 1

Sodium sulfate (Na^OJ

38 - 48 % I

Sodium perborate tetrahydrate

8-14%

TAED

0-3 %

CMC

0-3 %

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001-0.1

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

| Bulk density (g/1) at least

500 |

17) A detergent composition formulated as granulate

Soap, Na-salt

1-3 %

Nonionic, (eg. alcohol ethoxylate C, 2 _ 1B , 3-10 EO)

2-6%

Betaine (e.g Alkylamidopropylbetaine)

0-3 %

r nospnate (as o 1 r r)

27 - 37 % |

Sodium carbonate (Na 2 C0 3 )

17-23 %

Sodium disillicate; (NajO^SiOJ

3-7 %

Sodium sulfate (Na^O^

4- 11 %

Sodium perborate tetrahydrate

15 - 21 %

TAED

1-4%

CMC

1-3 %

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001-0.1

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

Bulk density (g/1) at least

600 1

18) A detergent composition formulated as granulate

Alkyl benzene sulfonic acid

5-11 %

Soap, Na-salt

0-3 %

Nonionic, (eg. alcohol ethoxylate C 12 . u , 3-10 EO)

3-7 %

Zeolite (NaAlSi0 4 ) (e.g Zeolite 4A)

20-30 %

Sodium carbonate (Na 2 CO,)

15-23 %

Sodium citrate /citric acid
(C^NajO/QHsO,)

0-3 %

Sodium sulfate (NajSO^

4-10%

Sodium percarbonate

7-13% I

TAED

2-6 %

CMC

1-3 %

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001-0.1

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

1 Bulk density (g/1) at least

600 1

19) A detergent composition formulated as granulate

Alkyl benzene sulfonic acid

0-4 %

Soap, Na-salt

0-3 %

Nonionic, (eg. alcohol ethoxylate C I2 _, 8 , 3-10 EO)

2-6%

Zeolite (NaAlSi0 4 ) (e.g Zeolite 4A)

11 - 17 %

Phosphate (as STPP)

25 - 35 % .

Sodium carbonate (N^COj)

3-7%

Sodium sillicate; (N^OiSiOJ

0-19%

Sodium sulfate (Na 2 S0 4 )

20 - 28 %

Sodium perborate tetrahydrate

9- 13 %

TAED

1-5%

CMC

0-2%

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001 -0.1

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

Bulk density (g/1) at least

600

20) A detergent composition formulated as granulate

Alkyl benzene sulfonic acid

17 - 23 %

Soap, Na-salt

0-3 %

Nonionic, (eg. alcohol ethoxylate C,^, 3-10 EO)

11 -15 %

Zeolite (NaAlSi0 4 ) (e.g Zeolite 4A)

60-70%

Sodium carbonate (NajCO,)

0-3%

Sodium sulfate (Na^O*)

5-11 %

CMC

0-3 %

Polymers (e.g PVP, PEG, maleic/acrylic acid
copolymer))

2-6%

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001-0.1

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

| Bulk density (g/1) at least

350

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

21) A detergent composition formulated as granulate

AlkyI benzene sulfonic acid

16 - 22 %

Soap, Na-salt

0-2 %

Nomomc, (eg. alcohol ethoxylate C 12l8 , 3-10 EO)

3-9 %

Zeolite (NaAlSiOJ

25 - 33 %

Sodium carbonate (Na 2 C0 3 )

3-7 %

Sodium sillicate; (NajOrSiOj)

0-4 %

Sodium sulfate (Na^C^)

5-11 %

rnuspnonaic

0-3 %

Sodium perborate monohydrate

15 - 19 %

TAED

3-7 %

CMC

0-3 %

Polymers (e.g PVP, PEG, maleic/acrylic acid
copolymer))

0-3 %

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001 - 0.1

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

Bulk density (g/I) at least

700

22) A detergent composition formulated as granulate

AlkyI benzene sulfonic acid

4-8 %

AlkyI sulfate (e.g C n . lg )

0-3 %

Nonionic, (eg. alcohol ethoxylate C„.„, 3-10 EO)

5-9 %

Zeolite (NaAlSi0 4 )

20 - 28 %

Sodium carbonate (N^CO,)

9-15 %

Sodium disillicate; (Na 2 0:2SiOj)

0-4 %

Sodium perborate tetrahydrate

21-31% I

TAED

1 -5 % I

CMC

0-3 % 1

Polymers (e.g PVP, PEG, maleic/acrylic acid
copolymer))

0-3 %

Enzymes including modified lipolytic enzymes
j (calculated as pure enzyme protein)

0.0001-0.1

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96700322

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

Bulk density (g/1) at least

600

23) A detergent composition formulated as a granulate comprising

Linear alkylbenzenesulfonate (calculated as acid)

6 - 12%

Nonionic surfactant

1 - 4%

Soap as fatty acid

2 - 6%

Sodium carbonate (as Na 2 C0 3 )

14 - 22%

Zeolite (as NaAlSi0 4 )

18 - 32%

Sodium sulfate (as Na^O^

5 -20%

Sodium citrate (as QHjNajOy)

3 - 8%

Sodium perborate (as NaBOj.H 2 0)

4 - 9%

Bleach activator (e.g. NOBS or TAED)

1 - 5%

Carboxymethylcellulose

0 - 2%

Polymers (e.g. polycarboxylate or PEG)

1 - 5%

Enzymes (calculated as pure enzyme
protein)

0.0001 - 0.1%

Minor ingredients (e.g. optical brightener, perfume)

0 - 5%

24) An aqueous liquid detergent composition comprising

linear alkylbenzenesulfonate (calculated as acid)

15-23%

Alcohol ethoxysulfate (e.g. C 12 IS
alcohol, 2-3 EO)

8 -15%

Alcohol ethoxylate (e.g. C 12 _ I5 alcohol, 7 EO, or
C l2 . u alcohol, 5 EO)

3-9% I

Soap as fatty acid (e.g. lauric acid)

0 - 3%

Aminoethanol

1 - 5%

Sodium citrate

5 -10%

Hydrotrope (e.g. sodium toluensulfonate)

2 - 6%

Borate (as B^)

0 - 2%

Carboxymethylcellulose

0-1% |

Ethanol

1-3% J

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96700322

Propylene glycol

2 - 5%

Enzymes (calculated as pure enzyme protein)

0.0001 - 0.1%

Minor ingredients (e.g. polymers, dispersants,
perfume, optical brighteners)

0 - 5%

25) An aqueous liquid detergent composition comprising

Linear alkylbenzenesulfonate (calculated as acid)

20 - 32%

Alcohol ethoxylate (e.g. C 12 . 15 alcohol,
7 EO, or C I2 . 1$ alcohol, 5 EO)

6 - 12%

Aminoethanol

2 - 6%

Citric acid

8 - 14%

Borate (as B^)

1 - 3%

Polymer (e.g. maleic/acrylic acid copolymer,
anchoring polymer such as, e.g., lauryl
methacrylate/acrylic arid copolymer)

0 - 3%

Glycerol

3 - 8%

Enzymes (calculated as pure enzyme
protein)

0.0001 - 0.1%

Minor ingredients (e.g. hydrotropes, dispersants,
perfume, optical brighteners)

0 - 5%

26) A detergent composition formulated as concentrated liquid.

Alkyl benzene sulfonic arid

6- 12 %

Alkyl sulfuric arid

0-4 %

Monoethanolamin

2-6%

Nonionic, (eg. alcohol ethoxylate C U . IS , 3-10 EO)

9- 15 %

1 Sodium citrate /citric arid
(QH^%0/CAO,)

2-8 %

Glycerol

2-6 % I

Borate (as Na^A)

0-4 % I

Polymers (e.g PVP, PEG, maleic/acrylic arid
copolymer))

0-3% I

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001 -0.1

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

Total water

40 %

27)

A detergent composition formulated as concentrated liquid.

Alkyl benzene sulfonic acid

11-17% j

Alkyl ether sulfuric acid (e.g. C 12 . 18 alcohol, 4-10
EO) or alkyl sulfate (e.g C„. 18 )

0-4 %

Triethanolamin

0-3 %

Nonionic, (eg. alcohol ethoxylate C l2 ., s , 3-10 EO)

6 - 10 %

Sodium citrate /citric acid
(C.H^CVQHsOt)

2-6%

Hydro tropes (Sodium toluene sulfonate)

1 -5 %

Glycerol

6 - 12 % |

MPG

0-5 %

Ethanol

0-3 %

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001 -0.1

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

Total water

55 %

5 28) A detergent composition formulated as a granulate having a bulk density of at least
600 g/1 comprising

| Anionic surfactant (linear alkylbenzenesulfonate,
| alkyl sulfate, alpha-olefinsulfonate, alpha-sulfo fatty
acid methyl esters, alkanesulfonates, soap)

25 -40%

Nonionic surfactant (e.g. alcohol
ethoxylate)

1 -10%

Sodium carbonate (as Na^O,)

8 -25%

Soluble silicates (as NaA ISiO^)

5 -15%

Sodium sulfete (as NajSO^

0 - 5%

Zeolite (as NaAlSi0 4 )

15 - 28%

Sodium perborate (as NaBO,.4H 2 0)

0 -20%

Bleach activator (TAED or NOBS)

0 - 5%

Enzymes (calculated as pure enzyme
protein)

0.0001- 0.1%

Minor ingredients (e.g. perfume, optical

0 - 3%

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

| brighteners)

29) A detergent composition formulated as granulate

Alkyl benzene sulfonic acid

25 - 35 % |

Noruonic, (eg. Alcohol ethoxylate C I2 ., 8 , 3-10 EO)

0-3%

Zeolite (NaAlSi0 4 )

3-9 %

Phosphate (as STOP)

25 - 35 %

Sodium carbonate (Na^COj)

0-3 %

Sodium dKilliratp* fNFn fV9SIin 1

Z - O TV

Sodium sulfate fNa*SCM

17 - 9^ PL

Sodium perborate monohydrate

1-5%

TAED

0-3 %

CMC

0-3 %

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001-0.1

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5%.

Bulk density (g/1) at least

600 J

30) A detergent composition formulated as granulate

| Alkyl benzene sulfonic acid

25 - 35 % 1

Soap, fatty acid Na-salt

0-3%

Nonionic, (eg. alcohol ethoxylate C^.,5 , 7 EO)

4-9 %

Zeolite (NaAlSi0 4 )

7-11%

Phosphate (as STPP)

26-36%

Sodium carbonate (NajCO,)

6 - 12 % 1

Sodium disillicate; (Na 2 0:2Si0 2 )

4 - 10 %

1 Sodium sulfate (Na^SOJ

4-8 %

CMC

0-3 %

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001 - 0.1

Minor ingrediens (e.g suds supressors, perfume,
optical brightener, photobleach)

0-5 %

Bulk density (g/1) at least

700

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

The following specific compositions are meant to exemplify compositions for the present
invention, but are not necessarily meant to limit or otherwise define the scope of the
invention.

In the detergent compositions, the abbreviated component identifications have the
following meanings:

LAS: Sodium linear C 12 alkyl benzene sulphonate
TAS: Sodium tallow alkyl sulphate
XYAS: Sodium C 1X - C iy alkyl sulfate

SS: Secondary soap surfactant of formula 2-butyl octanoic acid

25EY: A C, 2 - C I5 predominantly linear primary alcohol condensed with an average

of Y moles of ethylene oxide
45EY: A C I4 - C l5 predominantly linear primary alcohol condensed with an average

of Y moles of ethylene oxide
XYEZS: C 1X - C 1Y sodium alkyl sulfate condensed with an average of Z moles of

ethylene oxide per mole
Nonionic: C u - C 15 mixed ethoxylated/propoxylated fatty alcohol with an average

degree of ethoxylation of 3.8 and an average degree of propoxylation of 4.5

sold under the tradename Plurafax LF404 by BASF GmbH
CFAA: C 12 - C, 4 alkyl N-methyl glucamide
TFAA: C I6 - C l8 alkyl N-methyl glucamide
Silicate: Amorphous Sodium Silicate (Si0 2 :Na 2 0 ratio = 2.0)
NaSKS-6: Crystalline layered silicate of formula d-Na^O,
Carbonate: Anhydrous sodium carbonate
Phosphate: Sodium tripolyphosphate

MA/AA: Copolymer of 1:4 maleic/acrylic acid, average molecular weight about
80,000

Polyacrylate: Polyacrylate homopolymer with an average molecular weight of 8,000 sold

under the tradename PA30 by BASF GmbH
Zeolite A: Hydrated Sodium Aluminosilicate of formula Na^AlOjSiO^. 27H 2 0

having a primary particle size in the range from 1 to 10 micrometers
Citrate: Tri-sodium citrate dihydrate
Citric: Citric Acid

Perborate: Anhydrous sodium perborate monohydrate bleach, empirical formula
NaBCVHA

PB4: Anhydrous sodium perborate tetrahydrate

Percarbonate: Anhydrous sodium percarbonate bleach of empirical formula
2Na 2 C0 3 .3H 2 0 2

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

TAED: Tetraacetyl ethylene diamine
CMC: Sodium carboxymethyl cellulose

DETPMP: Diethylene triamine penla (methylene phosphonic acid), marketed by

Monsanto under the Tradename Dequest 2060
PVP: Polyvinyl pyrrolidone polymer

EDDS: Ethylenediamine -N, N'- disuccinic acid, [S,S] isomer in the form of the
sodium salt

Suds Suppressor:25% paraffin wax Mpt 50°C, 17% hydrophobic silica, 58% paraffin oil

Granular Suds: 12% Silicone/silica, 18% stearyl alcohol, 70% starch in granular form

Sulphate: Anhydrous sodium sulphate

HMWPEO: High molecular weight polyethylene oxide

TAE 25: Tallow alcohol ethoxylate (25)

Composition 1

A granular fabric cleaning composition in accordance with the invention may be prepared
as follows:

Sodium linear C, 2 alkyl

6.5

benzene sulfonate

Sodium sulfate

15.0

Zeolite A

26.0

Sodium nitrilotriacetate

5.0

Enzyme of the invention

0.1

PVP

0.5

TAED

3.0

Boric acid

4.0

Perborate

18.0

Phenol sulphonate

0.1

Minors

Up to 100

Composition 2

A compact granular fabric cleaning composition (density 800/1) in accord with the
invention may be prepared as follows:

45AS 8.0
25E3S 2.0
25E5 3.0

SUBSTITUTE SHEET (RULE 26)

WO 97/04079 PCT/DK96/00322

3.0

2.5
17.0
12.0
3.0
7.0
5.0
0.4
0.1
6.0
22.0
0.3
3.5

Up to 100%

• Composition 3

Granular fabric cleaning compositions in accordance with the invention which are
especially useful in the laundering of coloured fabrics were prepared as follows:

I n

20 LAS 10.7

TAS 2.4

TFAA - 4.0

45AS 3.1 10.0

45E7 4.0

25 25E3S - 3.0

68E11 1.8

25E5 - 8.0

Citrate 15.0 7.0
Carbonate - 10

30 Citric acid 2.5 3.0

Zeolite A 32.1 25.0

Na-SKS-6 - 9.0

MA/AA 5.0 5.0

DETPMP 0.2 0.8

35 Enzyme of the invention 0.10 0.05

Silicate 2.5

Sulphate 5.2 3.0

25E3
TFAA
Zeolite A
NaSKS-6
5 Citric acid
Carbonate
MA/AA
CMC

Enzyme of the invention
io TAED

Percarbonate
EDDS

Granular suds suppressor
water/minors

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

PVP

Poly (4-vinylpyridine)-N-
Oxide/copolymer of vinyl
imidazole and vinyl-
pyrrolidone
Perborate
Phenol sulfonate
Water/Minors

Composition 4

Granular fabric cleaning compositions in accordance with the invention which provide
"Softening through the wash" capability may be prepared as follows:

45AS

10.0

LAS

7.6

68AS

1.3

45E7

4.0

25E3

5.0

Coco-alkyl-dimethyl hydroxy-

1.4

1.0

ethyl ammonium chloride

Citrate

5.0

3.0

Na-SKS-6

11.0

Zeolite A

15.0

MA/AA

4.0

DETPMP

0.4

Perborate

15.0

Percarbonate

15.0

TAED

5.0

Smectite clay

10.0

HMWPEO

0.1

Enzyme of the invention

0.10

0.05

Silicate

3.0

5.0

Carbonate

10.0

Granular suds suppressor

1.0

4.0

CMC

0.2

0.1

Water/Minors

Up to 100%

0.5

0.2

1.0
0.2

Up to 100%

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

Composition 5

Heavy duty liquid fabric cleaning compositions in accordance with the invention may be
prepared as follows:

LAS acid form

Citric acid

25AS acid form

2SAE2S acid form

25AE7

CFAA

DETPMP

Fatty acid

Oleic acid

Ethanol

Propanediol

Enzyme of the invention

Coco-alkyl dimethyl

hydroxy ethyl ammonium

chloride

Smectite clay

PVP

Water / Minors

5.0
8.0
3.0
8.0
5

1.0

4.0
2.0
0.10

25.0
2.0

1.0

6.0

0.05

3.0

5.0

2.0

Up to 100%

Composition 6

A detergent composition formulated as a granulate

Alcohol ethoxylate C„.„, 5-7 EO

Alkyl benzene sulfonate; C„., 3

Linear alkyl sulfate; C 16 .„
(e.g Sulfopon)

Soap

4 |

Sodium carbonate

Zeolith Na-A

Sodium sillicate; Na 2 0:Si0 2 =3

2 I

Sodium sulfate (NajS0 4 )

20 1

Polycarboxylate; (Sokalan- CP5)

5 I

Carboxylic acids (Sokalan DCS)

4 |

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

oLKii ui ii pcruuniie leuanyurate

TAED

Suds supressors

Enzymes including modified lipolytic
enzymes (calculated as pure enzyme
protein)

0.0001 -0.1

Composition 7

A bleach containing detergent composition formulated as a granulate

| Primary alcohol sulfate (CocoPAS)

R Nonion 3EO (e.g Alkohol ethoylate C12-
15; 3EO)

Nonion 7EO (Alkohol ethoylate C12-15:
7EO)

Sokalan HP22*

0,7

0,8

Soap

1,3

Zeolite MAP

Sodium citrate

Sodium carbonate

3,3

Water/ salts

0,4

0,5

Antifoam/flourescer/perfume

TAED

Mn-catalyst

1,7

1,7 I

Percarbonate

21 |

EDTMP

0,4

Enzymes including modified lipolytic
enzymes (calculated as pure enzyme
protein)

0.0001-0.1

0.0001 -0.1

• a graft polymer as decribed in WO 95/22593

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

Cpmposition g

A non-bleach detergent composition formulated as a granulate

Primary alcohol sulfate (CocoPAS)

Nonion 3EO (e.g Alkohol ethoylate

Nnninn TFO (f* 0 AllmHrJ t*ihrwr}nte*

C12-15; 7EO)

1 A
14

Sokalan HP22*

0.7

0.6

0.7

0.5

Soap

Zeolite MAP

Sodium citrate

Sodium carbonate

Sodium CMC

0.7

Water/ salts

Antifoam/P VP/perfume

EDTMP

1.4

Enzymes including modified lipolytic
J enzymes (calculated as pure enzyme
] protein)

0.0001-0.1

• A graft polymer as decribed in the invention W095622593
Composition 9

A determent compositio n formulated as a granulate

A detergent composition formulated as a
granulateA detergent composition
formulated as a granulateAlcohol ethoylat
C„. u , 5EO

0.5

Alkyl glycoside,C, J . M . Degree of
polymerization: 1,4

Linear alkyl sulfate C l6 .„ (e.g Sulfopon,
Henkel)

Soap

Sodium carbonate

Zeolith Na-A

33 1

SUBSTITUTE SHEET

WO 97/04079 PCT/DK96/00322
81

Sodium silicate (SiC^N^O = 3.0)

Polycarboxylate (Sokalan CP5)

Carboxylic acids (Sokalan DCS)

Sodium percarbonate

TAED

Sud supressors

Enzymes including modified lipolytic
enzymes calculated as pure enzyme
protein)

0.0001 -0.1

Water

to 100 |

Composition 10

A detergent composition formulated as a granulate

Zeolite A

Sodium disiucate (e.g SKS-6, Hoechst)

Amorphes sodium silicate
NaiOiSiOj = 1:2.0

5.5

Sodium carbonate

Polycarboxylate (Sokalan CP5)

Mixt. Of 80% Alcohol ethoxylate C„ „, 5
EO and 20 % Alcohol ethoxylate C t2 ., 4 , 3
EO (Dehydol LST 80/20)

2.5

Alkohol ethoxylate C 16 .„; 14 EO (e.g
Dehydol TA 14)

Sodium alkyl benzene sulfonate; C, 2

Alkyl sulfate; C l6 . lg

7.5

5.5

7.5

Perborate tetrahydrate

TAED

Enzymes including modified lipolytic
enzymes (calculated as pure enzyme
protein)

0.0001-0.1

1 Water, perfume, sud supressor ect.

To 100 |

Composition 1 1

A detergent composition formulated as a granulate

linear alkylbenzene sulfonate

& — i

Nonionic ethoxylated alcohol 7EO - (e.g

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

1 oynperonic a / )

I PtUIlIUIUl* CLUUAjidlCU diGUnUl, 1.1 IIilAl. UI

Superionic A3 & A7 (3 / 7 EO groups)

Zeolite 4A

Poly carboxylate (e.g Sokalan CP 5)

Sodium carbonate

Granular sodium silicate

TAED

Sodium perborate monohydrate

EDTMP (Ethylene diamine tetramethylene
phosphonic acid)

0.4

Nonionic material containing at least 25
EO groups (e.g Lutensol AT - BASF and
BRD- ICI)

0-1

Minor ingrediens (Fluoresces CMC, salts
antifoam granules, perfume

Enzymes including modified lipolytic
enzymes (calculated as pure enzyme
protein)

0.0001-0.1

Moisture

to 100

Composition 12

A detergent composition formulated as a granulate

Sodium primary alkyl sulphate (PAS)

Nonionic ethoxylated alcohol 3EO - (e.g
Synperonic A3)

Nonionic ethoxylated alcohol 7EO - (e.g
Synperonic A7)

Zeolite MAP

Stearic acid

Tallow 80EO

0,2

Sodium silicate

3 |

TAED

H Manganese catalyst

2 |

| Sodium percarbonate

21 |

| Dequest 2047

0,4

1 Minor ingrediens (Fluorescer, CMC, salts

4 I

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

| antifoam granules, perfume)

Enzymes including modified lipolytic
enzymes (calculated as pure enzyme
protein)

0.0001 -0.1 1

Moisture

to 100

Composition 13

A detergent composition formulated as a granulate

Decylidene or dodecylidene diglycerol

Decylidene or dodecylidene triglycerol

C12-15E07ethoxyIate

Zeolite

Sodium carbonate

Alkaline sodium silicate

Fatty acid soap

Sodium carboxy methyl cellulose

Sodium perborate monohydrate

TAED

Bleach stabiliser (EDTMP)

0.4

Silicone suds suppressor

0.4

flourescer/perfume

Enzymes including modified lipolytic
enzymes (calculated as pure enzyme
| protein)

0.0001-0.1

| Moisture To 100

Comoosition U

A detergent composition formulated as a granulate

| Alkyl benzene sulfonate C,o. u

Linear alkyl sulfate; C 1W , (e.g Sulfopon)

Alcohol ethoxylate; C 16 . u ; 5 EO

1.5

0.5

Cetyl/oleyl alcohol 5 EO

1.5

0.7

1.4

Cetyl/oleyl alcohol 10 EO

1.5

0.7

1.4

Alkohol ethoxylate C I4 . 15 ; 7 EO (Dobanol
45-7)

0.5 J

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

Alkohol ethoxylate C,4., 5 ; 4 EO (Dobanol
45-4)

0.5

Zeolith Na-A

Na-silicate

Na-caibonate

fl Na-sulfate

| Polycarboxylate (Sokalan CPS)

I Na-hydrogensulfate

1 Org. acid (Sokalan DCS)

1.5

Water

14.5

11.8

10.1

14.5

16.0

13.1

Soap; C„. 1R

Enzymes including modified lipolytic
enzymes (calculated as pure enzyme
protein)

0.0001-0.1

Composition 15

A powdered detergent composition

Sodium Alkylbenzene sulfonate C lM3

11.5

Alcohol ethoxy sulfate (Sulfated Alfonic
1412-70)

5.5

Primary alkohol sulfate (

Alcohol Ethoxylate (e.g Neodol 25-9)

Soap

R Sodium tripolyphosphate

| Aluminosilicates, e.g Zeolite 4A

10-
35

0-15

5-20

0-12

Polycarboxylate (e.g. CPS)

0-3

MA/AA/Hydrophobe terpolymers *

2-25

2-20

Alkaline Silicate

2-5

3-20

Sodium carbonate

Quaternary amines

2.4

Ethoxylated Amine (e.g Varonic U202)

SUBSTITUTE SHFR

WO 97/04079

PCI7DK96/00322

owcuiiig way

Flourescers (Tinopal AMS)

0.15

0.2

0.25

0.15

1.5

Perfume

0.1

0.2

0.1

Enzymes including modified lipolytic
enzymes (calculated as pure enzyme
protein)

0.0001 -0.1

Sodium sulfate

to balance |

* As described in US 5,308,530
Composition \6

An aqueous liquid washing agent

1 Alkyl benzene sulfonate; C l(M3 ;
] Monoethanolaminsalt

Alkyl ether sulphate, C 12 . I4 ;2EO

Sodium lauryl sulfate

Soap (C J2 . ir fatty acid)

Alkohol ethoxylate C 12 _ u ; 7EO

Alkylglycoside, C^- degree of
oligomerization: 1,6

AIkylglycoside,C l2 . ltf - degree of
oligomerization: 1,4

1,2-propanediol

Ethanol

Enzymes including modified lipolytic
enzymes (calculated as pure enzyme
protein)

0.0001-

0.1

Water

TolOO |

Composition 17

A liquid detergent composition

| Fattyacid monoglyceride (e.g Cutina AGS, Henkel)

0.5

| C n . 1B fatty acid (e.g Edenor K 12-18, Henkel)

Alkohol ethoxylate C I2 ., a ; 7EO

Alkylglycoside, C 12 . M ,degree of polymerization:
1.4

Alkyl sulfate C 1Ma (e.g Sulfopon K35, Henkel)

5 j

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

Ethanol

1 2 oroovlene plvcol

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001-0.1

Water

to 100

Composition 18

A detergent composition formulated as non-aqueous liquid detergent

| Alkohol ethoxylate C10-12; 7EO

| Alkohol ethoxylate C10-12; 3EO

Glycerol triacetate

Silicone antifoam

1.5

Alkv I benzene sulnhonic acid

* mm mm w m. mm ^rmm^r iflill/llvlliV 11t rlliF

Sodium carbonate

Calcite

Antiseeding polymer

Silica

Carboxy methyl cellulose

Peracids*

0-1

Brightener

0.2

Perfume

0.6

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001-0.1

* As described in WO 95/06104

Composition 19

A detergent composition formulated as a non aqueous liquid detergent:

Decylidene or dodecylidene diglycerol

C10-15EO 7 ethoxylate

Sodium carbonate

Sodium perborate monohydrate

Alkylbenzene sulphonic arid

Calcium carbonate

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

Silica (dispersant)

Silicone suds suppressor

flourescer/ antiashing polymer/antiredeposition
polymer

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001-0.1

Composition 20

A detergent composition formulated as aqueous Iiq. Detergents

Decyl- or dodecylidene triglycerol

Decyl- or dodecylidene diglycerol

12.5

C 10 .!5 EO 7, ethoxylate

12.5

Fatty acid

4.5

Potassium hydroxide

Zeolite

Citric acid

Glycerol

Borax

1.5

Polymer

Silicone oil

0.3

Perfume

0.5

Enzymes including modified lipolytic
enzymes (calculated as pure enzyme
protein)

0.0001-0.1

Water

to 100

Composition 21

A liquid detergent composition comprising

| Sodium C11-C15 Alkylbenzene sulfonate

| Alcohol ethoxy sulfate (C12-14, 60%
H ethylene oxide by weight)

I Alcohol Ethoxylate (12-14C
H alcohol)ethoxylate

B Alkylpolyglycoside

jj Trisodium citrate

0-15

0-10

0-20

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

Carboxymethylenoxysuccinate, tri sodium

0-20

Oxysuccinate - tetrasodium

MA/ AA/Hydrophobe terpolymers *

5-15

2-20

2-15

1-10

2-15

Monoethanoiamine

0-4

Triethanolamine

Sodium carbonate

Borax pentahydrate

3.5

Glycerol

Propylene glycol

Formic acid

Calcium chloride

Quaternary amines

1 Ethoxylated Amine

J Alkyldimethyl Amine Oxide

1.5

I Na Xylene Sulfonates

3 .

Ethanol

e
o

Flourescers

0.25

0.2

0.25

0.2

0.15

Perfume

0.2

0.15

0.1-
0.3

0.2

0.25

0.1-
0,25

Enzymes including modified lipolytic
enzymes (calculated as pure enzyme
protein)

0.0001-0.1

« y

Sodium sulfate

to balance Q

• As described in the invention US 5,308,530

SUBSTITUTE SHEP

WO 97/04079

PCI7DK96/00322

Composition 22

An aqueous liquid detergent composition

Silicon antifoam

0.3

Citric acid

Glycerol

i —

Borax

KOH

Zeolite 4A

Polymer Deflocculating polymer w. chemical
structure as polymer All in EP 346,995

1.0

QCC 200 : Bentonite clay

Oleic acid

LAS-acid

Synperonic A3

Synperonic A7

PVP

0.3

Perfume

0.5

Enzymes including modified lipolytic enzymes
(calculated as pure enzyme protein)

0.0001-0.1

Water

To 100

Dishwashing Composition

The dishwashing detergent composition comprises a surfactant which may be anionic, non-
ionic, cationic, amphoteric or a mixture of these types. The detergent will contain 0-90% of
non-ionic surfactant such as low- to non-foaming ethoxylated propoxylated straight-chain
alcohols.

The detergent composition may contain detergent builder salts of inorganic and/or organic
types. The detergent builders may be subdivided into phosphorus-containing and non-phos-
phorus-containing types. The detergent composition usually contains 1-90% of detergent
builders.

Examples of phosphorus-containing inorganic alkaline detergent builders, when present,
include the water-soluble salts especially alkali metal pyrophosphates, orthophosphates, po-
lyphosphates, and phosphorates. Examples of non-phosphorus-containing inorganic builders,

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

when present, include water-soluble alkali metal carbonates, borates and silicates as well as
the various types of water-insoluble crystalline or amorphous alumino silicates of which
zeolites are the best-known representatives.

Examples of suitable organic builders include the alkali metal, ammonium and substituted
ammonium, citrates, succinates, malonates, fatty acid sulphonates, caiboxymetoxy succinates,
ammonium polyacetates, carboxylates, polycarboxylates, aminopolycaiboxylates, polyacetyl
caiboxylates and polyhydroxsulphonates.

Other suitable organic builders include the higher molecular weight polymers and co-polymers
known to have builder properties, for example appropriate polyaoylic acid, polymaleic and
polyacrylic/polymaldc acid copolymers and their salts.

The dishwashing detergent composition may contain bleaching agents of die
chlorine/bromine-type or the oxygen-type. Examples of inorganic chlorine/bromine-type
bleaches are lithium, sodium or calcium hypochlorite and hypobromite as well as chlorinated
trisodium phosphate. Examples of organic chlorine/bromine-type bleaches are heterocyclic N-
bromo and N-chloro imides such as trichloroisocyanuric, tribromoisocyanuric, dibromo-
isocyanuric and dichloroisocyanuric acids, and salts thereof with water-solubilizing cations
such as potassium and sodium. Hydantoin compounds arc also suitable.

The oxygen bleaches are preferred, for example in the form of an inorganic persalt,
preferably with a bleach precursor or as a peroxy acid compound. Typical examples of
suitable peroxy bleach compounds are alkali metal perborates, both tetrahydrates and
monohydrates, alkali metal percarbonates, persilicates and perphosphates. Preferred activator
materials are TAED and glycerol triacetate.

The dishwashing detergent composition of the invention may be stabilized using conventional
stabilizing agents for the enzyme(s), e.g. a polyol such as e.g.propylene glycol, a sugar or a
sugar alcohol, lactic add, boric add, or a boric add derivative, e.g. an aromatic borate ester.

The dishwashing detergent composition may also comprise other enzymes, in particular an
amylase, a protease and/or a cellulase.

The dishwashing detergent composition of the invention may also contain other conventional
detergent ingredients, e.g. deflocculant material, filler material, foam depressors, anti-cor-
rosion agents, soil-suspending agents, sequestering agents, anti-soil redepositkm agents,

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

dehydrating agents, dyes, bactericides, fluoresces, thickeners and perfumes.

The first wash lipolytic enzyme of the invention may be incorporated in concentrations
conventionally employed in detergents. It is at present contemplated that, in the detergent
composition of the invention, the lipolytic enzyme may be added in an amount corresponding
to 0.00001-1 mg (calculated as pure enzyme protein) of lipolytic enzyme per liter of wash
liquor.

Below, specifically preferred diswashing compositions are exemplified: .

1) POWDER AUTOMATIC DISHWASHING COMPOSITION

Nonionic surfactant

0.4 -2.5%

Sodium metasilicate

0 -20%

Sodium rfisiticate

3 -20%

Sodium triphosphate

20 - 40%

Sodium carbonate

0 -20%

Sodium perborate

2 - 9%

Tetraacetyl ethylene diamine (TAED)

1 - 4%

Sodium sulphate

5 -33%

Enzymes

0.0001 -0.1%

2) POWDER AUTOMATIC DISHWASHING COMPOSITION

| Nonionic surfactant
(e.g. alcohol ethoxylate)

1 - 2%

Sodium disilicate

2 -30%

Sodium carbonate

10 -50%

Sodium phosphonate

0 - 5%

Trisodium citrate dihydrate

9 -30%

hfitrilotrisodium acetate (NTA)

0 -20%

Sodium perborate monohydrate

5 - 10%

Tetraacetyl ethylene diamine (TAED)

1 - 2%

Polyacrylate polymer

(e.g. maleic acid/acrylic acid copolymer)

6 -25%

Enzymes

0.0001 - 0.1%

SUBSTITUTE SHEET

WO 97/04079

PCIYDK96/00322

Perfume

0.1 - 0.5%

Water

5 -10

3) POWDER AUTOMATIC DISHWASHING COMPOSITION

Nonionic surfactant

0.5 - 2.0% |

Sodium disilicate

25 -40%

Sodium citrate

30 - 55%

Sodium carbonate

0 -29%

Sodium bicarbonate

0 -20%

Sodium perborate monohydrate

0 - 15%

Tetraacetyl ethylene diamine (TAED)

0 - 6%

Maleic acid/acrylic
acid copolymer

0 - 5%

day

1 - 3%

Polyamiiro acids

0 -20%

Sodium polyacrylate

0 - 8%

Enzymes

0.0001- 0.1%

4) POWDER AUTOMATIC DISHWASHING COMPOSITION

Nonionic surfactant

1 - 2%

Zeolite MAP

15 -42%

Sodium disilicate

30 - 34%

Sodium citrate

0 -12%

Sodium carbonate

0 -20%

Sodium perborate monohydrate

7 -15%

Tetraacetyl ethylene
diamine CTAED)

0 - 3%

Polymer

0 - 4%

Maleic acid/acrylic acid copolymer

0 - 5%

| Organic phosphorate

0 - 4%

1 - 2%

Enzymes

0.0001-0.1%

Sodium sulphate

Balance

SUBSTITUTE SHEET (RULE 26)

WO 97/04079

PCT/DK96/00322

5) POWDER AUTOMATIC DISHWASHING COMPOSITION

Nonionic surfactant

I - 7%

Sodium disilicate

18 -30%

Trisodium citrate

10 - 24%

Sodium carbonate

12 - 20%

Monopersulphate (2 KHSOs.KHSO^K^OJ

15 -21%

Bleach stabilizer

0.1 - 2%

Maleic acid/acrylic acid copolymer

0 - 6%

Diethylene triamine pentaacetate,
pentasodium salt

0 - 2.5%

Enzymes

0.0001 - 0.1%

Sodium sulphate, water

Balance |

6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANIN
SURFACTANT SYSTEM

1 Nonionic surfactant

0 - 1.5%

Octadecyl dimethylamine N-oxide dihydrate

0 - 5%

80:20 WLC18/C16 blend of octadecyl dimethylamine
N-oxide dihydrate and hexadecyldimethyl amine N-
oxide dihydrate

0 - 4%

70:30 wtC18/C16 blend of octadecyl bis
(hydroxyethyl)amine N-oxide anhydrous and
hexadecylbis

(hydroxyethyl)amine N-oxide anhydrous

0 - 5%

Cu-C u alkyl ethoxysulfate with an average degree of
ethoxylation of 3

0 -10%

Cu-Ctf alkyl ethoxysulfate with an average degree of
ethoxylation of 3

0 - 5%

C^-Cji ethoxylated alcohol with an average degree of
ethoxylation of 12

0 - 5%

A blend of C^-C^ ethoxylated alcohols with an
average degree of ethoxylation of 9

0 - 6.5%

A blend of C 13 -C l5 ethoxylated alcohols with an
average degree of ethoxylation of 30

0 - 4%

Sodium disilicate

0 -33% I

Sodium tripolyphosphate

0 -46% 1

SUBSTITUTE SHEET

WO 97/04079

PCT7DK96/00322

Sodium citrate

0 -28%

Citric acid

0 -29%

Sodium carbonate

0 -20%

Sodium perborate monohydrate

0 -11.5%

Tetraacetyl ethylene diamine (TAED)

0 - 4%

Maleic acid/acrylic acid copolymer

0 - 7.5%

Sodium sulphate

0 - 12.5%

Enzymes

0.0001 - 0.1%

7) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING COMPOSITION

liquid nonionic surfactant (e.g. alcohol ethoxylates)

2.0 - 10.0%

Alkali metal silicate

3.0 - 15.0%

Alkali metal phosphate

20.0 -40.0%

liquid carrier selected from higher

glycols, polyglycols, polyoxides, glycolethers

25.0 -45.0%

Stabilizer (e.g. a partial ester of phosphoric acid and a
C 16 -C, 8 alkanol)

0.5 - 7.0%

Foam suppressor (e.g. silicone)

0 - 1.5%

Enzymes

0.0001 - 0.1%

8) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION

liquid nonionic surfactant (e.g. alcohol ethoxylates)

2.0 - 10.0%

Sodium silicate

3.0 - 15.0% I

Alkali metal carbonate

7.0 -20.0%

Sodium citrate

0.0 - 1.5%

Stabilizing system (e.g. mixtures of finely divided
silicone and low molecular weight dialkyl polyglycol
ethers)

0.5 - 7.0%

Low molecule weight polyacrylate polymer

5.0 - 15.0%

Clay gel thickener (e.g. bentonite)

0.0 - 10.0%

Hydroxypropyl cellulose polymer

0.0 - 0.6%

Enzymes

SUBSTITUTE SHEET

0.0001 - 0.1% |

WO 97/04079

PCT/DK96/00322

liquid carrier selected from higher lycols,
polyglycols, polyoxides and glycol ethers

Balance

9) THKOTROPIC UQUID AUTOMATIC DISHWASHING COMPOSITION

12^^14 laJXy aCIQ

0 - 0.5%

Block co-polymer surfactant

1.5 - 15.0%

Sodium citrate

0 -12%

Sodium tripolyphosphate

0 -15%

Sodium carbonate

0 - 8%

Aluminium tristearate

0 - 0.1%

Sodium cumene sulphonate

0 - 1.7%

Polyacrylate thickener

1.32 - 2.5%

Sodium polyacrylate

2.4 - 6.0%

Boric add

0 - 4.0%

Sodium formate

0 - 0.45%

Calcium formate

0 -0.2%

Sodium n-decydiphenyl oxide disulphonate

0 - 4.0%

Monoethanol amine (MEA)

0 - 1.86%

Sodium hydroxide (50%)

1.9 - 9.3%

1,2-Propanediol

0 - 9.4%

Enzymes

0.0001 - 0.1%

Suds suppressor, dye, perfumes, water

Balance

10) UQUID AUTOMATIC DISHWASHING COMPOSITION

Alcohol ethoxylate

0 -20%

Fatty add ester sulphonate

0 -30%

Sodium dodecyl sulphate

0 -20%

ADcyl polyglycoside

0 -21% |

Oleic add

0 -10%

Sodium di silicate monohydzate

18 -33%

Sodium dtrate dihydrate

18 - 33%

Sodium steaiate

0 - 2.5% J

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

Sodium perborate monohydrate

0 -13%

Tetraacetyl ethylene diamine (TAED)

0 - 8%

Maleic arid/acrylic add copolymer

4 - 8%

Enzymes

0.0001 - 0.1%

11) LIQUID AUTOMATIC DISHWASHING COMPOSITION CONTAINING
PROTECTED BLEACH PARTICLES

Sodium silicate

5 -10%

Tetrapotassium pyrophosphate

15 -25%

Sodium triphosphate

0 - 2%

Potassium carbonate

4 - 8%

Protected bleach particles, e.g. chlorine

5 -10%

Polymeric thickener

0.7 - 1.5%

Potassium hydroxide

0 - 2%

Enzymes

0.0001 - 0.1%

Water

Balance

11) Automatic dishwashing compositions as described in 1), 2), 3), 4), 6) and 10), wherein
perborate is replaced by percaibonate.

12) Automatic dishwashing compositions as described in 1) - 6) which additionally contain a
manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described in
"Efficient manganese catalysts for low-temperature bleaching Nature 369 r 1994, pp. 637-
639.

Furthermore, the first wash lipolytic enzyme of the invention may be used in softening
compoaticms:

The lipolytic enzyme of the invention may be used in fabric softeners, e.g. as described in
Surfactant and Consumer Products, Ed. by J. Falbe, 1987, pp 295-296; Tenside Surfactants
Detergents, 20 (1993), 6, pp 394-399; JAOCS, Vol. £1 (1984), 2, pp 367-376; EP 517 762;
EP 123 400; WO 92/19714; WO 93/19147; US 5,082,578; EP 494 769; EP 544 493; EP
543 562; US 5,235,082; EP 568 297; EP 570 237.

Finally the invention relates to a process for washing or cleaning different subject matters
using a composition of the invention. By using said composition it is possible more effectively

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

to remove lipid deposits from said subject matters clothes, textiles or dishes.

It is also possible to reduce the amount of lipolytic enzymes which must be present in the
detergent composition to obtain the same washing performance as detergent compositions
comprising the parent lipolytic enzyme.

In an embodiment of the invention the process comprises the following steps:

a) soaking the subject matter in aqueous medium,

b) contacting for a period of time the subject matter to a detergent composition comprising the
modified enzyme with lipolytic activity of the invention dissolved in the aqueous medium
before, during or after step a),

c) rinsing the subject matter,

d) removing the rinsing water from the subject matter.

SUBSTITUTE SHEET

WO 97/04079

PCJ7DK96/00322

MATERIALS AND METHODS
MATERIALS

Plasmids:

pYES 2.0 (Invitrogen Corp., UK)

p960 A oryzae expression plasmid (described in EP 305 216 from Novo Nordisk A/S)
10 pSX581 (R colt expression plasmid) (see figure 7)

pJS037 (S. cerevisiae expression plasmid) (Okkels J.S., Annals of the New York Academy of

Sciences (in press 1995) (see also figure 8)

pSX167 (see figure 4)

pSX92 (WO 89/06279)
is pUC19 (Yanish-Penon et al. (1985) Gene 33, 103-119)

pHD414 (Aspergillus expression vector being a derivative of the plasmid p775 described in

EP 238 023). The construction of pHD414 is further described in WO 93/11249).

PJVi245 (See Figure 9)

pCaHj383 (see Figure 9)

25 pCaHj385 (see Figure 9)

pAHE2: Hobson, A. H., Buckley, C. M., Aamand, J. L., Jergensen, S. T., Dide-
richsen, B., and McConnell, D. J. (1993). Activation of a bacterial lipase by its chapero-
30 ne. Proc. NaU. Acad. Sci. USA, 90, p. 5682-5686).

35 Microorganisms:

Saccharomyces cerevisiae YNG318: MATa Dpep4[cir + ] ura3-52, ku2-D2, his 4-539

SUBSTITUTE SHEET

WO 97/04079

PCI7DK96/00322

Aspergillus oryzae IFO 4177

A. oryzae JaL 125: Aspergillus oryzae IFO 4177 available from Institute for Fer-
mention, Osaka; 17-25 Juso Hammachi 2-Chome Yodogawa-ku, Osaka, Japan, having the
alkaline protease gene named tt alp" (described by Murakami K et aL, (1991), Agric. Biol.
5 Chem. 55, p. 2807-2811) deleted by a one step gene replacement method (described by G.
May in "Applied Molecular Genetics of Filamentous Fungi" (1992), p. 1-25. Eds. J. R.
Kinghorn and G. Turner; Blackie Academic and Professional), using the A. oryzae pyrG gene
as marker.

E a>tfW31101acF(£ coli W31 10 is an early isolate used as ancestral stock for the K-12
10 strain (Bachman, (1972), Bacteriol. Rev. 36). The W3 110 slain has been made lacP in order

to overproduce the Lac repressor, turning off expression from plac more completely.

£. coli SJ6: Diderichsen, B., Wedsted, U., Hedegaard, L., Jensen, B. R., Sjeholm,

C, (1990), Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoen-

zyme from Bacillus brevis. J. Bacteriol., 172, p. 4315-4321).
15 Strain SJ1503 is E. coli JA221 containing plasmid pAHE2:

Hobson, A. H., Buckley, C. M., Aamand, J. L., Jergensen, S. T., Diderichsen, B., and

McConnell, D. J. (1993). Activation of a bacterial lipase by its chaperone. Proc. Natl.

Acad. Sci. USA, 90, p. 5682-5686.

Donor organisms:

Humicola lanuginosa DSM 4109 (EP 305,216)
Humicola insolens DSM 1800 (WO 96/13580)
25 Pseudomonas cepacia SB10, DSM 3959, is described in WO 89/01032.

Enzymes:

Bovine trypan (Boehringer Mannheim)

SUBSTITUTE SHEET

WO 97/04079

PCT/DK96/00322

100

The following lipases are variants of the Humicola lanuginosa DSM 4109 lipase (EP 305 216)
which are either used as parent enzymes in the context of the present invention or which con-
stitute modified enzymes according to the invention.

5 Table Ml

Lipase van*
ants

Peptide
addition

Mutations

ITT vie

SPIRR

D57G, N94K, D96L, L97M

HLvl

D57G, N94K, D96L, L97M

HLv2s

SPIRR

D137G, D167G, E210V, W221L

HLv2

DI37G, D167G, E210V, W221L

HLv3s

SPIRR

N94K, F95L.1396H, N101S, F181L, D234Y, I252L,
P256T, G263A, L264Q

HLv3

N94K, F95L, D96H, N101S, F181L, D234Y, E52L,
P256T, G263A, L264Q

HLv4s

SPIRR

I90F, D96L, E99K, V187A

HLv4

I90F, D96L, E99K, V187A

HLvds

SPIRR

N94K, D96A, Q249R

TTT ,_C

NbWK, D96A, Q249R

TTT ./T-

HLV/S

SPIRR

D57G, G59V, N94K, D96L, L97M, S116P, SI70P,
Q249R

HLv7

D57G, G59V, N94K, D96L, L97M, S116P, S170P,
Q249R

HLv8s

SPIRR

A49P, D167G, E210V

HLvg

A49P, D167G, E210V

HLv9s

SPIRPRP

D57G N94K, D96L, Q249R

HLv9

D57G N94K, D96L, Q249R

HLvlOsl

GPIRPRP

D57G, N94K, D96L, L97M, Q249R

HLvl0s2

SHSRHNA

D57G, N94K, D96L, L97M, Q249R

HLvl0s3

TAIRPRK

D57G, N94K, D96L, L97M, Q249R

HLvl0s4

SALRRRP

D57G, N94K, D96L, L97M, Q249R

HLvl0s5

STRRPRP

D57G, N94K, D96L, L97M, Q249R

SUBSTITUTE SHEET

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PCT/DK96/00322

101

TJT trlrtr^

2>PRRPRT

D57G, N94K, D96L, L97M, Q249R

TTT - - 1 f\—*l

HLvlOsv

SPIPPGP

D57G, N94K, D96L, L97M, Q249R

X1L.V1US5

D57G, N94K, D96L, L97M, Q249R

HLvl0s9

SPFRPKL

D57G N94K D96I TQ7M CftdQV

^-'-'/Vlj l^^TXV, i/7UL»j

HLvlOslO

SALRRP

D57G, N94K, D96L, L97M, Q249R

HLvlOsll

SPIRK

D57G, N94K, D96L, L97M, Q249R

HLvl0sl2

SPIR

D57G, N94K, D96L, L97M, Q249R

HLvlO

D57G, N94K, D96L, L97M, Q249R

HLvlls

SPIRP

E1P, D57G, N94K, D96L, L97M, Q249R

The following lipases are variants of the B. cepacia (formerly Pseudomonas cepacia) lipase to
which an N-terminal addition has been applied in accordance with the present invention.

5 Table M2

Lipase vari-
ants

Peptide
addition

SJ3708

SPIRR

SJ3717

SPIRPRP

SJ3718

SPIRPRP

SJ3719

TAIRPRK

SJ3720

STRRPRP

SJ3720

STRRPRP

SJ3721

GPIRPRP

The following lipases are variants of the Hwnicola insolens DSM 1800 lipolytic enzyme.

Table M3

Lipase vari-
ants

Peptide
addition

HILvls

SPPRRP

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PCT/DK96/00322

102

HILV2s

SPPRP

HILv3s

SPIRK

HBLv4s

PPPRRPR

Enzyme inhibitor;

Soy bean trypsin inhibitor (Boehringer Mannheim)
Media:

5 YPD: 10 g yeast extract, 20 g peptone, H 2 0 to 810 ml. Autoclaved, 90 ml 20% glucose
(sterile filtered) added.

LB-medium: 10 g Bacto-tryptone, 5 g Bacto yeast extract, 10 g NaCl in 1 litre water.

FG4 medium: 3% soy meal, 3% maltodextrin, 1% peptone, pH adjusted to 7.0 with 4 M

NaOH

10 Litex Agarose HSB 2000 (CAT NO: F90472)

BG-reagent: 4 mg/ml Brilliant Green (BG) dissolved in water
Substrate 1:

10 ml Olive oil (Sigma CAT NO. 0-1500)
20 ml 2% polyvinyl alcohol (PVA)
15 The Substrate is homogenised for 15-20 minutes.
PCS detergent
10 gfi:

SDS 0.52 g

D6banol25-3 0.60 g

20 Dobanol25-7 0.58 g

NaBOjHjO 1.50 g

Ad 1 litre 0.1 M Tris buffer (pH 9), and dilute further with the Tris buffer to the double
concentration of the desired concentration on the PCS plates.
PCS-plates
25 Solution for making PCS plates

RECTIFIED SHEET (RULE 91)

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103

Brilliant Green (BG-reagent) 10 ml

Substrate 1 24 ml

PCS detergent 500 ml

2% agarose CmTRIS buffer (pH 9) 500 ml
5 Lipase Substrate (Sigma catalogue no. 800-1)
Brilliant Green (Merck, art. No. 1.01310)
Swatches:

3.5 x 3.5 cm and 9x9 cm cotton swatches (style #400 from TestFabrics, Inc. (New Jersey)
stained with lard/sudan red
to Ljinl: Lard coloured with 0.75 mg sudan red/gram lard.

Detergent I:

1.17 g/1 LAS (Nansa 1169/P, 30% a.m.)
0.15 g/1 AEO (Dobanol 25-7)
15 1.25 g/1 Sodium triphosphate
LOO g/1 Sodium sulphate
0.45 g/1 Sodium carbonate
0.15 g/1 Sodium silicate
The pH adjusted to 10

Inactivated Ariel Futur (Procter and Gamble) (commercially available batch No.4279 B
23:35): The enzymes in the detergent were inactivated by heat (4 minutes at
85°C in miaooven).

25 Water 3.2 mMCa^/Mg 2 ^ (5:1)

Chameleon double-stranded, site directed mutagenesis kit (caL no. 200509) (Stratagene, La-
joUe, CA)

30 Eouipment:

473A Protein Sequencer (Applied Biosystems)

SUBSTITUTE SHEET

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PCI7DK96/00322

104

Toyopearl Butyl column (XK 16/10) (Pharmacia, Sweden)
Q-Sepharose column (HPQ XK 26/10) (Pharmacia, Sweden)
MonoQ column (1 ml) (Pharmacia, Sweden)
Highperformance Q SeparoseO (Pharmacia, Sweden)
5 SpinlOO column (Clontech Lab. Inc., CA, USA)

METHODS:

Hybridization conditions

Medium to high stringency
Presoaking in 5X SSC and prehydbridizing for 1 hour at about 40°C in a solution of 20%
formamide, SX Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 mg denatured
15 sonicated calf thymus DNA, followed by hybridization in the same solution supplemented
with 100 mM ATP for 18 hours at about 40°C, followed by a wash in 0.4X SSC at a tempe-
rature of about 45°C.

Construction of veast expression vector
20 The expression plasmid pIS037, is derived from pYES 2.0. The inducible GAL1-
promoter of pYES 2.0 was replaced with the constitutively expressed TPI (triose phosphate
isomerase)-promoter from Saccharomyces cerevisiae (Albert and Karwasaki, (1982), J. MoL
Appl GeneL, 1, 419-434), and the URA3 promoter has been deleted. A restriction map of
pJS037 is shown in figure 8.

Meftpd for constructing lipolytic variant

The peptide addition and/or mutations in the non-structural N-terminal and/or C-terminal
end of the parent lipolytic enzyme to construct modified lipolytic enzymes of the invention
were performed either by site-directed mutagenesis or by random mutagenesis.

Site-directed mutagenesis

SUBSTITUTE SHEET

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105

For the construction of variants of a H. lamiginosa lipolytic enzyme the commercial kit,
Chameleon double-stranded, site-directed mutagenesis kit can be used according to the manu-
facturer's instructions.

The gene encoding the lipolytic enzyme in question is inserted into the plasmid pHD414.

5 In accordance with the manufacturer's instructions the Seal site of the Ampicillin gene of

pHD414 is changed to a Mlul site by use of the following primer

Primer 3: AGAAATCGGGTATCCTTTCAG (SEQ ID No. 6)

The pHD414 vector comprising the lipolytic gene in question is then used as a template

for DNA polymerase and oligos 7258 and 7770. The desired mutation (e.g. in the N-terminal

10 of the lipolytic gene) is introduced into the lipolytic gene in question by addition of an appro-

. priate oligos comprising the desired mutation.

PGR reactions are performed according to the manufacturer's recomendations.

Random mutagenesis

is May be performed essentially as described in WO 95/22615. More specifically, for per-
forming random mutagenesis in short DNA stretches such as in the peptide addition, the ran-
dom mutagenesis is performed by use of doped or spiked oligonucleotide probes. For larger
DNA stretches PCR generated mutagenesis may be used.

20 Low calcium filter assay
Procedure

1) Provide SC Ura" replica plates (useful for selecting strains carrying an expression vector)
with a first protein binding filter (Nylon membrane) and a second low protein binding filter
(Cellulose acetate) on the top.
25 2) Spread yeast cells containing a parent lipase gene or a mutated lipase gene on the double
filter and incubate for 2 or 3 days at 30°C.

3) Keep the colonies on the top filter by transferring the topfilter to a new plate.

4) Remove the protein binding filter to an empty petri dish.

5) Pour an agarose solution comprising an olive oil emulsion (2% P.V.A.:01ive oil =3:1),
30 Brilliant green 0ndicator,O.OO4%), 100 mM tris buffer pH9 and EGTA (final concentration

5mM) on die bottom filter so as to identify colonies expressing lipase activity in the form of
blue-green spots.

SUBSTITUTE SHEET

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106

6) Identify colonies found in step 5) having a reduced dependency for calcium as compared to
the parent lipase.

Dobanol®25-7 filter assay :
5 The screening for an improved tolerance towards a detergent component is performed by use
of a filter assay corresponding to that described above except for the fact that the solution
defined in 5) further comprises 0.02% Dobanol®25-7 and optionally without any EGTA.

An alternative screening a<t<ay is th e following:
10 Procedure

1) Provide SC Ura-plates (useful for selecting strains carrying an expression vector) with a
protein binding filter (Cellulose acetate) on the top.

2) Spread yeast ceils containing a parent lipase gene or a mutated lipase gene on the filter and
incubate for 3 or 4 days at 30°C.

15 3) Keep the colonies on the top filter by transferring the topfilter to a new plate.
4) Remove the protein binding filter to a petri dish containing:

An agarose solution comprising an olive oil emulsion (2% P.V.A/.Olive oil=2:l), Brilliant
green (indicator,0.004%), 100 mM tris buffer pHIO and the detergent or detergent
component, e.g. PCS-plates.
20 5) Identify colonies expressing lipase activity in the form of blue-green spots found in step 4)

Fermentation in yeast

10 ml of SC-ura medium are inoculated with a S. cerevisiae colony and grown at 30°C
for 2 days. The resulting 10 ml culture is used for inoculating a shake flask containing 300 ml
25 SC-ura* medium which is grown at 30°C for 3 days. The 300 ml is used for inoculation 5 litre
of the following G-substrate:

400 g

Amicase

6.7 g

yeast extract (Difco)

12.5 g

L-Leucin (Fluka)

30 6.7 g

10 g

MgS0 4 7H,0

SUBSTITUTE SHEET

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107

17 g

K^0 4

Tracecompounds
Vitamin solution

10 ml

Sml

6.7 ml

H,P0 4

20% Plutonic (antifoam)

5 25 ml

In a total volume of 5000 ml:

The yeast cells are fermented for 5 days at 30°C. They are given a start dosage of 100 ml
70% glucose and added 400 ml 70% glucose/day. A pH=5.0 is kept by addition of a 10%
NH, solution. Agitation is 300 rpm for the first 22 hours followed by 900 rpm for the rest of
io the fermentation. Air is given with 11 air/l/min for the first 22 hours followed by 1.5 1
air/l/min for the rest of the fermentation.

Trace compounds: 6.8 g of ZnCl,. 54.0 g of FeCl^O, 19.1 g of MnO^HA 2.2 g of
CuSO^O, 2.58 g of CaCt 0.62 g of H 3 BO, , 0.024 g of (NHJ 6 MoA,4HA 0.2 g of
H, 100 ml of HQ (concentrated), in a total volume of 1 1.
is Vitamin solution; 2 50 mg of ffiotin, 3 g of Thiamin, 10 g of D-Calciumpanthetonate, 100 g
ofMyo-mositol.50gofCholiiichlorid, 1.6 g of Pyridoxin, 1.2 g of Niacinainid, 0.4 g of
Folicacid, 0.4 g of Riboflavin. In a total volume of 1 litre.

Transformation of Aspergillus orvzae (general procedure)

20 100 ml of YPD (Sherman et al., (1981), Methods in Yeast Genetics, Cold Spring Harbor
Laboratory) are inoculated with spores of A. oryzae and incubated with shaking for about 24
hours. The mycelium is harvested by filtration through miracloth and washed with 200 ml of
0.6 M MgS0 4 . The mycelium is suspended in 15 ml of 1.2 M MgS0 4 , 10 mM NaHjPO*, pH
5.8. The suspension is cooled on ice and 1 ml of buffer containing 120 mg of Novozym* 234,

25 batch 1687 is added. After 5 min., 1 ml of 12 mg/ml BSA (Sigma type H25) is added and
incubation with gentle agitation continued for 1.5-2.5 hours at 37°C until a large number of
protoplasts is visible in a sample inspected under the microscope.

The suspension is filtered through miracloth, the filtrate transferred to a sterile tube and
overlayed with 5 ml of 0.6 M sorbitol, 100 mM Tris-HCl, pH 7.0. Centrifugation is per-

30 formed for 15 min. at 1000 g and the protoplasts are collected from the top of the MgS0 4
cushion. 2 volumes of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM CaCy are

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added to the protoplast suspension and the mixture is centrifugated for 5 min. at 1000 g. The
protoplast pellet is resuspended in 3 ml of STC and repelleted. This is repeated. Finally, the
protoplasts are resuspended in 0.2-1 ml of STC.

100 pi of protoplast suspension are mixed with 5-25 y.g of p3SR2 (an A. nididans amdS

5 gene carrying plasmid described in Hynes et al., Mol. and Cel. BioL, Vol. 3, No. 8, 1430-
1439, Aug. 1983) in 10 fd of STC. The mixture is left at room temperature for 25 min. 0.2
ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl 2 and 10 mM Tris-HCl, pH 7.5 is added
and carefully mixed (twice) and finally 0.85 ml of the same solution are added and carefully
mixed. Hie mixture is left at room temperature for 25 min., spun at 2.500 g for 15 min. and

10 the pellet is resuspended in 2 ml of 1.2M sorbitol. After one more sedimentation the pro-
toplasts are spread on minimal plates (Cove, (1966), Biochem. Biophys. Acta 113, 51-56)
containing 1.0 M sucrose, pH 7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to
inhibit background growth. After incubation for 4-7 days at 37°C spores are picked, suspen-
ded in sterile water and spread for single colonies. This procedure is repeated and spores of a

is single colony after the second re-isolation are stored as a defined transfbrmanL

Fed batch fermentation

Fed batch fermentation is performed in a medium comprising maltodextrin as a carbon
source, urea as a nitrogen source and yeast extract The fed batch fermentation was performed

20 by inoculating a shake flask culture of A oryzae host cells in question into a medium compri-
sing 3.5% of the carbon source and 0.5% of the nitrogen source. After 24 hours of cultivation
at pH 5.0 and 34°C the continuous supply of additional carbon and nitrogen sources are initi-
ated. The carbon source is kept as the limiting factor and it is secured that oxygen is present in
excess. The fed batch cultivation is continued for 4 days, after which the enzymes can be re-

25 covered by centrifugation, ultrafiltration, clear filtration and germ filtration. Further purifica-
tion may be done by amonexchange chromatographic methods known in die art

Lipase acuity (LU - Lipase Units)

Lipase activity is assayed using glycerine tributyrate as a substrate and gum-arabic as an
30 emulsifier. 1 LU (lipase Unit) is the amount of enzyme which liberates 1 pmol titratable

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butyric acid per minute at 30°C, pH 7.0. The lipase activity is assayed by pH-stat using
Radiometer titrator VIT90, Radiometer, Copenhagen.

Application of lard on the swatches
5 6 ml of stained lard heated to 70°C are applied to the earner of each swatch. After ap-
plication of the stain the swatches are heated in an oven for 25 minutes at 75°C and stored
overnight at room temperature prior to the first wash.

3-cvcle wash performance

10 The 3-cycle wash performance of a modified lipolytic enzyme of the invention can be
evaluated on the basis of the enzyme dosage in mg of protein (or LU) per litre compared to
the parent lipolytic enzyme. Wash trials 2re carried out in 150 ml beakers placed in a ther-
mostated water bath. The beakers are stirred with triangular magnetic rods.
The experimental conditions are as follows:

is Method: 3 cycles with overnight drying between each cycle
Wash liquor: 100 ml per beaker

Swatches: 6 swatches (3.5 x 3.5 cm, stained with lard coloured with 0.75 mg sudan

red/gram of lard) per beaker

Detergent: Detergent I, pH adjusted to 10.2
20 Enzyme cone: 0.075, 0.188, 0.375 f 0.75 and 2.5 mg of lipase protein per litre

Time: 20 minutes

Temperature: 30°C

Rinse: 15 minutes in running tap water

Drying: overnight at room temperature ("20°C, 30-50% RH)

25 Evaluation: after the 3rd wash, the reflectance at 460 nm was measured.

Evaluations of wash results

Dose-response curves are compared for the modified lipolytic enzyme and the parent
lipolytic enzyme. The dose-response curves is calculated by fitting the measured data to the
30 following equation:

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DR = DR_ (I)

K + C 05

5 where DR is the effect expressed in reflectance units
C is the enzyme concentration (mg/1)
DIL is a constant expressing the maximum effect

K is a constant; K 2 expresses the enzyme concentration at which half of the ma-
ximum effect is obtained.

Based on the characteristic constants DR^ and K found for each modified lipolytic en-
zyme as well as the parent lipolytic enzyme, improvement factors are calculated. The impro-
vement factor, defined as

= c^c (n)

expresses the amount of modified lipase protein needed to obtain the same effect as that obtai-
ned with 0.25 mg/1 of the reference parent protein (C^J.

Thus, the procedure for calculating the improvement factor is as follows:
20 1) The effect of the parent protein at 0.25 mg/1 (DI^J was calculated by means of equation

(i);

2) the concentration of the modified lipolytic enzyme resulting in the same effect as the parent
enzyme at 0.25 mg/1 was calculated by means of the following equation:

25 c = dU) ) 2 on)

3) the improvement factor was calculated by means of equation (II).

30 1 cycle wash performance

1 cycle wash trials are carried out in a termostated Terg-Oto-Meter (TOM).

Method: 1 cycle wash followed by linedrying.

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Wash liquor: 1000 ml per beater

Swatches: 7 swatches (9x9 cm, stained with lard coloured with 0.75 mg sudan red/gram
of lard)

Water: 3.2 mM Ca^/Mg 2 * (5:1)
5 Detergent: 5 g/1 inactivated Ariel Futur*. Natural pH around 10.3. (commercially availa-
ble batch No.4279 B 23:35).

Lipase concentrations: 0, 1250, 12500 LU/1

Time: 20 minutes

Temperature: 30°C
io Rinse: 15 minutes in running tap water.

Drying: Overnight at room temperature (' 20°C f 30-40 % RH).

Evaluation: The fatly matter is extracted using the soxhlet method and the amount of
fatty matter is gravimetrically determined.

15 EXAMPLES
EXAMPLE 1

Production of wjldtvoe Hundcola lanuginosa lipase m veast

For expression Humicola lanuginosa lipase in the yeast Saccharomyces cerevisiae

20 YNG318 the yeast expression vector pJS037 (see figure 8) was constructed as described in
the Material and Methods section above. pJS037 comprises the DNA sequence encoding
the parent lipase and includes the DNA sequences encoding the signal peptide and propepti-
de (see figure 1). The plasmid was transformed into the yeast by standard methods (cf.
Sambrooks et al. t (1989). Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring

25 Harbor). The yeast was cultivated as described in the Material and Methods section above.

Pn ^eation of H. lanurinosa lipas e expressed in ft cerevisiae

Ammonium acetate (92 g) was added to the fermentation broth (1400 ml) to give a 0.8 M
solution of ammonium acetate. The solution was added onto a Toyopearl Butyl column (XK
30 16/10). The column was washed wiih 0.8 M ammonium acetate and the lipase eluted in HjO
at a flow rate of 5 ml/min. 10 ml fractions were collected and lipase containing fractions
were pooled according to activity in the standard lipase titration assay. The lipase containing

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pooled according to activity in the standard lipase titration assay. The lipase containing pool
were filtered and the pH was adjusted to pH 7.6 and added onto a Q-Sepharose column (HPQ
XK 26/10). The column was washed with 200 ml 0.1 M Tris-HCl, pH 7.25 and the lipase
was eluted in a linear gradient of 0 to 0.3 M NaCl in 400 ml of 0.1 M Tris-HCl, pH 7.25 at a
5 flow rate of 5 ml/min. 10 ml fractions were collected and the lipase containing fractions were
pooled according to activity in the standard lipase titration assay. The lipase containing pool
was diluted with H 2 0 and added onto a 1 ml MonoQ column at a flow rate of 1 ml/min. The
column was washed with 30 ml of H 2 0 and the lipase was eluted in linear gradient of 0 to
0.25 M NaCl in 40 ml. The lipase was manually collected according to absorption at 280 nm.

N-terminal amino acid s equencing of H. lamtpinosa lipase expressed in veast

The N-terminal amino add sequencing was conducted on the S. cerevisiae expressed lipase
using the 473A Protein Sequencer according to the manufacturer's instructions.

When the N-terminal amino add sequence of 5. cerevisiae expressed lipase is compared to
15 the N-terminal amino add sequence of the same lipase expressed in A. oryzae (as described in
EP 305 216) a difference was observed, as the major part of the 5. cerevisiae expressed en-
zyme contains 5 amino add residues extra (SPERR-) at the N-terminus (see Table El) which
indudes the corresponding information for the A. oryzae expressed lipase.

Table El

Expression
system

Fraction containing
SPIRR-EVSQ...

Fraction containing
EVSQ...

£ cerevisiae

75 %

25%

A. oryzae

100%

As can be seen from the table a major portion of the secreted lipase expressed in 5. cerevi-
25 rice has been extended by the five amino acid SPIRR (from the pro-peptide). The relative

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amount of enzyme containing the extra amino acid residues can be established from the yi-
elds of PTH-amino acids in amino acid sequencing.

5 EXAMPLE 2

Rpmnval of the SPIRR-peptide from the N- te"ni™« o f the H. lanuginosa lipa se expressed
in S. cerevisiae

To 4.5 mg of the above purified modified ("SHRR"-contaiTring) lipase expressed in S.
10 cerevisiae (m 1.8 ml 0.05 M NH 4 HCOj) was added 50 mg bovine trypsin (sequencing gra-
de) and the mixture was incubated for 1 hour at 37 6 C. Upon incubation the tryptic digest
was stopped by adding more than 50 mg soy bean trypsin inhibitor.

The removal of the N-tenninal SPIRR-peptide addition was observed by N-terrninal ami-
no acid sequencing where the traction containing SPIRR was reduced from 75 % to 13 %
15 (See Table E2).

Table E2

Treatment

Fraction containing

SPIRR-EVSQ...

EVSQ.,..

Untreated

75 %

25 %

(i.e. modified lipase)

Trypsin

13 %

87%

treatment

The mild trypsin treatment did not result in internal cleavages in the modified lipase as
no internal amino acid sequences were observed by amino acid sequencing. Also the speci-
fic activity of the trypsin treated lipase was comparable to specific activity of the untreated
lipase shewing that the trypsin treatment did not affect enzyme activity in the standard assay
(See Table E3).

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Table E3

Sample

Amo

Activity
(LU/ml)

Specific Activity
(LU/AW

Untreated

(i.e. modified lipase)

2.5

1-8

9725

3890

Trypsin treated

2.2

1.8

9163

4127

5 EXAMPLE 3

Production of wildtvpe ft lanuginosa lipase in Aspergillus orvme

Cloning of Humicola lanuginosa lipase is described in EP 305,216, which also describes
expression and characterization of the lipase in Aspergillus oryzae by use of the expression
plasmid p960. Said plasmid comprises the Humicola lanuginosa lipase encoding gene and the

to DNA sequence encoding the SPIRR peptide addition (which is a part of the propeptide coding
part of the lipase gene). The expression of the wildtype lipase in A. oryzae (strain IFO 4177)
was done essentially as described in WO 95/22615 using an expression plasmid slightly modi-
fied as compared to p960 - cf WO 95/22615.

is Purification of wildtype H. lanuginosa lipase expressed in A. oryzae

Fermentation supernatant from Aspergillus oryzae IFO 4177 was centrifuged and cell de-
bris discarded. The supernatant was filtered though a 0.45 m millipore filter.

Then the supernatant was precipitated with 60% saturated ammonium sulphate. The preci-
pitate was dissolved in water and solid ammonium acetate was added to a final concentration
20 of 0.8 M. The solution was applied onto a Butyl Toyopearl column which was pre-
equOibrated with 0.8 M ammonium acetate. The bound enzyme was duted with gradient
using water and 50% ethanol as eluent

Fractions containing enzyme activity are then pooled and conductance was adjusted too it is
lower than 5 mSi and pH is adjusted to 7.5.

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The pools containing activity were then applied onto an anion exchange column (e.g.
Highperformance Q Separose®) which had been pre-equilibiated with 25 raM Tris-acetate
buffer, pH 7.5. The bound activity was eluted with linear salt gradient using same buffer and
0.5 M sodium chloride. Fractions containing high lipase activity were pooled.

EXAMPLE 4

Construction of pa rent Humicola lanuginosa lipase expression vector and expression in E. coli
pSX92 (see figure 4) was cut with Hind HI, blunt ended with Klenow polymerase and then
cut with Clal. The large fragment was isolated (A). pHLL (see EP 305,216 figure 3 and 4)
io (comprising the DNA sequence encoding the parent lipase) was cut with BamHl , blunt ended,
and cut with XhoII. The fragment containing the mature part of the modified lipase gene was
isolated (B).

A and B were ligated together with a synthetic linker (KFN 575/576) which codes for the
last 5 amino adds in the subtilisin 309 signal fused to the first four amino acids of die mature
is lipase. The last nucleotide "A" in the upper strand changed the XhoII site in the mature lipase
gene to a Bgl n ate.

Synthetic linker

KFN 575/576: 5'- CG ATCGC ATCGGCTGCTG AGGTCTCGC AA-3 '
20 3-TAGCGTAGCCGACGACTCCAGAGGCTTC^AG-5 ,

The resulting plasmid (pSX167) comprised the DNA sequence encoding die mature lipase.
pSX167 was cut with Pme I and Bam HI and the fragment containing the subtilisin 309 signal
sequence-lipase fusion and the 5S terminator was isolated (1769 bp). This fragment was liga-
25 ted into Hinc H-Bam HI cut pUC19 creating pSX578.

DNA coding for mature lipase down to Bst XI (from pSX167, 654 bp) was fused to the
Achromobacter tyticus protease I signal sequence (see figure 3) from Sph I using the PGR
technique 'Splicing by Overlap Extension - , Horton et al., (1989), Gene).

Plasmid (pSX578) (see figure 5) was cut with Sph I and Bst XI and the above mentioned
30 PGR DNA was inserted (figure 6). The resulting plasmid pSX581 (see figure 7) was trans-
formed into £. coli W31 10 lacP. When grown in shake flasks for 72 hours in LB-medium

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containing 0.4% lactose at 30°C the resulting strain produces non-glycosylated lipase with the
same specific activity as the normal glycosylated parent lipase enzyme.

EXAMPLE 5

5 Construction of H. lanuginosa lipase with peptide addition in K coli

The pSX581 plasmid (see figure 7) was digested with Bgin/Hindin and the vector frag-
ment was purified from an agarose gel using standard methods.
A PCR reaction was performed with the following primers using pSX581 as template:

io SPIRR primer Primer 1 tSEO ID NO 3^:

5'- AA CAG ATC TT G CGA GAC CTC TCT ACG TAT AGG GCT AGC GAG CGC
GGC GCT GAT CG -3' (55-mer)
PCR primer Primer 2 (SEP ID NO 4^:
GTTGTGTGGAATTGTGAGCGG (21-mer)

The resulting 300 bp fragment was purified on Spin 100 columns and digested with
Bgin/Hindm and again spin 100 purified. This fragment was ligated to the above vector frag-
ment. The resulting plasmid was named pJS0215 and used to transform Rcoli W3110 lacF.
A plasmid preparation was made from a transformant and DNA sequenced to verify the intro-
20 duction of the SPIRR peptide addition.

EXAMPLE 6

Construction and expression of modified H. lanuginosa lipolytic enzyme fHLv9s> in
Aspergillus orvzae JaL125

An N-terminal peptide addition was applied to the parent H. lanuginosa (DSM 4109)
lipolytic enzyme having the amino acid and DNA sequence, respectively, apparent from
EP 305 216, and in addition carrying the following mutations D57G, N94K* D96L,
Q249R in its mature part (inserted by conventional site-directed mutagenesis) in the DNA
30 sequence (EP 305 216). The peptide addition SPIRPRP was applied to the N-terminus of
the parent enzymes as follows:

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Construction of pIVI220:

The plasmid was constructed using the Chamelon double stranded, site-directed
mutagenesis kit from Stratagene according to the described protocol.
5 pHL296 was used as the plasmid template. Said plasmid contains the gene

encoding the K lanuginosa lipolytic enzyme with the above mentioned mutations (D57G,
N94K, D96L, L97M, Q249R) cloned into pHD464.

Primer no. 7258 was used as the selection primer.
7258: 5'p gaa tga ctt ggt tga cgc gtc acc agt cac 3* (SEQ ID NO. 79)
10 (Thus changing the Seal site found in the ampicillin resistance gene and used for cutting
to a Nflul site).

Primer no. 7770 was used as the selection primer.
7770: 5'p let age cca gaa tac tgg ate aaa tc 3 ' (SEQ ID NO. 80) (Changes the Seal site
found in the H. lanuginosa lipase gene without changing the amino acid sequence).
15 Primer no.8479 was used as the mutagenic primer.

8479: 5'p gcg tgg acg gec ttg get age cct att cgt cct cga ccg gtc teg cag gat ctg 3 (SEQ
ID NO 81) (replacing the propeptide and the N-terminal El of the parent H. lanuginosa
enzyme (SPIRRE by SPIRPRP).

20 Construction of pIVI245:

The plasmid was constructed using the Chameleon double-stranded, site-directed
mutagenesis kit from Stratagene (cat no. 200509) according to the described protocol.

pIVI220 was used as the plasmid templated and primer no.7887 as the selection
primer (changing the introduced Mlul site found in the ampicillin resistance gene and
25 used for cutting to a Seal site). 7887: 5'p-gaa tga ctt ggt tga gta etc acc agt cac 3" (SEQ
ID NO. 77).

Primer no. 8932 was used as the mutagenic primer (8932: 5'p-g aac tgg ata gga
aat ttg aag ttc ctg ttg aaa gaa ata aat gac 3' (SEQ ID NO. 78) (thus changing M97 back
to L97 as wildtype and still preserving the two mutations N94K and D96L)).

2. Construction of the A. orvzae expression plasmid pCaHj483

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pCaHj483 is depicted in Figure 9. It is build from the following fragments :

a) The vector pToC65 (W091/17243) cut with EcoRl and XbaL

b) A 2.7 kb Xbal fragment from A. nidulans carrying the amdS gene (C. M. Corrick et
ah, (1987), Gene 53, p. 63-71). The amdS gene is used as a selective marker in fungal

5 transformations. The amdS gene has been modified so that the BamHI site normally
present in the gene is destroyed. This has been done by introducing a silent point
mutation using Primer 3: AGAAATCGGGTATCCTTTCAG (SEQ ID No. 6)

c) A 0.6 kb EcoRII BamHI fragment carrying the A. niger NA2 promoter fused to a 60bp
DNA fragment of the sequence encoding the 5 * untranslated end of the mRNA of the A.

10 nidulans tpi gene. The NA2 promoter was isolated from the plasmid pNA2 (EP 383 779)
and fused to the 60 bp tpi sequence by PCR. The primer (Primer 4 SEQ ID No. 14)
encoding the 60 bp tpi sequence had the following sequence : 5'-
GCTCCTCATGGTGGATCCCCAGTTGTGTATATAGAGGATTGAGGAAGGAAGAG
AAGTGTGGATAGAGGTAAATTGAGTTGGAAACTCCAAGCATGGCATCCTTGC -

15 3'

d) A 675 bp Xbal fragment carrying the A. niger glucoamylase transcription terminator.
The fragment was isolated from the plasmid pICAMG/Term (EP 238 023).

The BamHI site of fragment c) was connected to the Xbal site in front bf the
transcription terminator on fragment d) via the pIC19R linker (BamHI to Xbal)

Construction of the HLv9s expression plasmid pCaHi485

The plasmid pTVi 245 was digested with BamH I and Sal I, and the resulting 904
bp fragment encoding the HLv9s lipolytic enzyme was isolated. pCaHj 483 was
digested with BamH I and Sal I, and the large vector fragment (6757) was ligated to the
25 HLv9s fragment The ligation mixture was used to transform E. colt DH5a cells, and a
transformant harbouring the expected plasmid was isolated. The plasmid was termed
pCaHj485.

3. Transformation of pCaHi 485 into JaL125
30 Aspergillus oryzae JaL 125 is Aspergillus oryzae IFO 4177 deleted in the alkaline

protease was transformed with pCaHj 485 using selection on acetamide as described in

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patent EP 0 531 372. Transformants were spore reisolated twice. Spores from second
reisolation of each transformant were used to inoculate 200 /d YPM (1% yeast extract,
2% peptone, 2% maltose) in 96 well microtiter dishes. The YPM cultures were grown
for 4 days at 34°C, and the higest producers were selected using a p-nitro phenylbutyrate
5 assay:

Stock solution: 18 /il p nitro phenyl butyrate was dissolved in 1 ml isopropanol.
Wprking solution: 0.1 ml stock solution was mixed with 10 ml 50 mM Tris/HCl pH 7.5;
10 mM Caa 2 .

Assay: 1 of YPM supernatant was mixed with 200 M I of working solution in 96 well
o microtiterdishes, and the color development was measured at 450 nm using an EUSA
reader.

One transformant was selected for tank fermentation.

4. Tank ferm entation of JaL 125/oCaHi 485

15 The fermentation was carried out as a fed-batch fermentation using a constant

medium temperature of 34°C and a start volume of 1.2 litre. The initial pH of the
medium was set to 6.5. Once the pH had increased to 7.0 this value was maintained
through addition of 10% H 3 P0 4 . The level of dissolved oxygen in the medium was
controlled by varying the agitation rate and using a fixed aeration rate of 1.0 litre air per

20 litre medium per minute. The feed addition rate was maintained at a constant level during
the entire fed-batch phase.

The batch medium contained maltose syrup as carbon source, urea and yeast
extract as nitrogen source and a mixture of trace metals and salts. The feed added
continuously during the fed-batch phase contained maltose syrup as carbon source

25 whereas yeast extract and urea were added in order to assure a sufficient supply of
nitrogen.

5. Purification nf the modified lipolytic enzyme

1) Fermentation supernatant was filtered through miliipore filter Cat No. AP2504700 Filter
30 type AP25.

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2) Fermentation supernatant was filtered once more on through the sterile filter from Millipore
membrane Type GS 0.22 micron.

3) Fermenation supernatent was then adjusted to 0.8 M ammonium acetate by adding solid
ammonium acetate,

5 4) A Hydrophobic chromatography on TSK gel Butyl-Toyopearl 650. 50 ml column was
packed with the Butyl-Toyopearl matrix. The column was washed and equilibrated with 0.8
M ammonium acetate. One liter ferementation supernatent adjusted with amonium acetate was
then applied on the Butyl column. The column was washed with 0.8 M ammonium acetate till
all unbound material was washed out. Bound material was then eluted with water and 50 %

to ethanol sequentially. Fractions were collected and analyzed for lipase activity using Standard
LU assay. Fractions containing lipase activity were pooled and diluted to adjust conductivity
of the pool below 4 mSi and pH to 8.5.

5) Anion exchange chromatography on High Performance Q sepharose (Pharmacia, Code
No. 17-1014-01). 50 ml column was packed and washed with 50 mM Borate buffer pH 8.5.
is Pool conatining lipase activity was then appUied on The High performance Q sepharose co-
lumn. Unbound material was washed with the Borate buffer pH 8.5. Bound activity was then
eluted with linear gradient using Borate buffer conatining 1 M Sodium Chloride pH 8.5.
Fractions were collected and assayed for lipase activity. Fractions containing lipase Activity
with a ratio of UV absorbence at A280and A260 more than 1.7 are pooled.

EXAMPLE 7

3-cvcle wash performance of H. lanuginosa lipase with a peptide addition

The wash performance of the Hwmcola lanuginosa lipase described in EP 305 216 and

variants thereof (i.e. modified lipolytic enzymes of the invention) was tested using the 3-cycle
25 wash performance test (described in die Materials and Methods section above) on swatches of

cotton soiled with lard stained with red colour in a model wash system at 30°C, at a constant

temperature for 20 minutes. The tests were performed at lipase concentrations of 0, 0.075,

0. 188, 0.375, 0.750, 2.500 mg enzyme protein per litre.

4.2 g/1 of a European type powder detergent composition (as described above) were used
30 in the test The detergent did not contain any enzymes prior to the addition of the modified

lipase of the invention. The detergent was dissolved in approximately 18°dH (German Hard-

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ness) water. The pH of the wash liquor was about 10- Six swatches were washed in 100 ml of
wash liquor. Subsequent to the washing, the swatches were flushed in running tap water for
15 minutes and then air-dried overnight at room temperature.
3 wash cycles were performed with overnight drying between each wash cycle.
5 After the third wash cycle the performance of a modified lipase of the invention and of the
parent lipase expressed in Aspergillus oryzae was assessed. This was done by calculating the
improvement factor (fimprove) as described above.

The results of these tests are shown in Table E4 below.

Table E4

Lipase

N-terminal
+/- SPIRR

3-cyclesf^
(Improvement factor)

Parent lipase
(expressed in A. oryzae)

1.0 (reference)

Modified lipase (expressed in yeast)

2.2

Modified lipase (expressed in yeast)
(treated with trypsin)

0.6

Variant of parent lipase (HLvls)
(expressed in yeast)

9.3

Variant of parent lipase (HLvl)
(expressed in A. oryzae)

1.8

Parent lipase (expressed in £ coli)

1.0

Modified lipase (expressed in £ coti)
(+SPIRR)

2.0

Modified lipase (expressed in Hansen-
Ida)

2.1

It can be seen from Table E4 that the peptide addition {i.e. SPIRR) applied to the N-terminal
of parent Humicola lanuginosa lipase at least doubles the wash performance.

EXAMPLE 8

One cycle wash performance of modified H. lanuginosa lipases containing an addition

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The one cycle wash performance test (described above in the Materials and Methods secti-
on above) was performed of Humicola lanuginosa lipase variants of Table Ml with and wit-
hout the SPIRR-peptide addition in 5 g/1 of enzyme inactivated ArieP Futur (Procter and
Gamble). The tests were performed at lipase concentrations of 0, 1250 12500 LU/1.
5 The detergent was dissolved in approximately 18°dH (German Hardness) water. The pH
of the wash liquor was about 10.3.

The amount of soxhlet extracted fatty matter removed from textile are shown in the table
below. Corresponding lipase variants with and without peptide addition are listed two and
two.

Table M5

Lipase vari-
ant

+/-
SPIRR

low dos-
age

% laid
removed

High dosage

% lard rem-
oved

HLv2s

SPIRR

1250 LU/1

12.5

12500 LU/1

HLv2

1250 LU/1

1.7

12500 LU/1

6.0

HLv3s

SPIRR

1250 LU/1

8.9

12500 LU/1

33.9

HLv3

1250 LU/1

4.6

12500 LU/1

6.9

HLv4s

SPIRR

2500 LU/1

26.5

12500 LU/1

47.6

HLv4

0.25 mg/1

12500 LU/1

HLvls

SPIRR

1250 LU/1

12.8

12500 LU/1

HLvl

1250 LU/1

1.8

12500 LU/1

7.2

HLv5s

SPIRR

1250 LU/1

11.4

12500 LU/1

36.5

HLv5

1250 LU/1

12500 LU/1

10.6

HLv8s

SPIRR

1250 LU/1

4.5

12500 LU/1

HLv8

1250 LU/1

12500 LU/1

nd: not determined

The above results clearly shows that the lipase variants with a peptide addition have a signifi-
15 cantly improved one cycle wash performance in comparison to the corresponding lipase vari-
ant without a peptide addition.

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EXAMPLE 9

Site-directed mutagenesis of N-terminal addition of H. lanuginosa lip ase

Mutations in the Hwnicola lanuginosa lipase having a SPIRR N-terminal addition was per-
formed using the method described above in the Materials and Methods section.
5 First the gene encoding the lipase was inserted into the plasmid pHD414. The Seal site of

the Ampicillin gene of pHD414 was then changed to a Mlul site. The unique Seal ate present

in the lipase gene was then removed.

The desired mutation (Le. SPIRPRP) was introduced in the N-terminal of the lipase gene

by addition of the following oligo comprising the desired mutation:
10 oligo 8479 (SEQ ID NO 5):

5'- P GCG TGG ACG GCC TTG GCT AGC CCT ATT CGT CCT CGA CCG GTC TCG

CAGGATCTG-3'

This resulted in a K lanuginosa lipase gene with a SPIRPRP N-terminal peptide addition.

15 EXAMPLE 10

Construction of N-terminal additions by random mutagenesis

Random mutagenesis of the part of the DNA sequence encoding the N-terminal addition

SPIRPRP added to the first amino acid residue of the mature H. lanuginosa lipolytic enzyme

(obtainable from DSM 4109) and containing the following further mutations in its mature
20 part D57G+N94K+D96L+L97M+Q249R was performed. The mutations in the mature

part of the parent lipolytic enzyme was performed by PCR driven site-directed mutagenesis

using the appropriate primer sequences using the procedures described in WO 95/26215 . The

peptide addition SPIRPRP was applied as described in Example 9.
The nucleotide doping scheme of the SPIRPRP codons was as follows:
25 Oligo 1: 5'- GCG TGG ACG GCC TTG GCC 86TT/A> 66f AID SZCT/X) 67n7A>

66(7/ A) SIS 66CTIM GAG GTC TCG CAG GAT CTG -3' (57-mer) (SEQ ID NO 82)

the numbers referring to which of the following flasks to be used.

Flask 5 : 80% A; 6,66% C; 6,66% G og 6,66 % T.
30 Flask 6 : 80% C; 6,66% A; 6,66% G og 6,66 % T.
Flask 7 : 80% G; 6,66% A; 6,66% C og 6,66 % T.

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Flask 8 : 80% T; 6,66% A; 6,66% C og 6,66 % G.

A two step PGR reaction protocol was used: The first step with the above primer as the
5* primer and with the primer 2056 (5'gca cgt aat gtt tgt acc 3 1 ) as the 3' primer con-

s ducted using pHL296 as the plasmid template. The product of the first PCR round was
used in a new PCR with 4699 (5'cgg lac ccg ggg ate cac 3*) as the 5* primer (to intro-
duce the BamHI site and the first part of the coding sequence) and with the PCR product
as the 3* primer using the same template. The resulting product was purified on Spin 100
(from Clonetech Lab., Inc.) and cut with BamHI and PvuII. The resulting DNA fragment

10 was purified from the agarose gel with SpinX (Costar) and ligated into the yeast expres-
sion vector pIS037 containing the H. lanuginosa lipolytic enzyme gene from pHL296
cloed as a BamHI-Xbal fragment cut with BamHI and PvuII. The resulting DNA was
electrotransformed into DH10/DH12 E. coli cells (Gibco/BRG Lifetechnologies) using
the conventional technique.

is After transformation into E. coli and amplification the plasmid was purified and transfor-
med into S. cerevisiae YNG 318. The resulting 5. cerevisiae cells were screened for good per-
formers in the alternative lipase filter assay containing detergent (3 g/1 of PCS). The positives
were sequenced and found to contain the peptide additions apparent from table 1 in ^Mate-
rials and Methods section (identified as HLvlOsl-10).

20 The one-cycle wash performance of each of HL10sl-6 was tested as described in the Mate-
rials and methods section above (One cycle wash performance) at a temperature 30°C and
using 5 gA of inactivated Arid Future as detergent. The amount of fatty material removed by
each of the modifed enzymes are shown below:

Lipase variant

Low dosage

% lard
removed

High dosage

% lard
removed

HLvlOsl

1250 LU/1

12500 LU/1

HLvl0s2

1250 LU/1

12500 LU/1

HLvl0s3

1250 LU/1

12500 LU/1

HLvl0s4

1250 LU/1

12500 LU/1

JhLvIOsS

1250 LU/1

12500 LUA

47 |

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HLvl0s6

1250 LU/1

12500 LU/1

53 '1

HLvlOsll

1250 LU/1

12500 LU/1

HLvl0sl2

12500 LU/1

15.8

HLvlls

1250 LU/1

12500 LU/1

The tendency was that the best performers had more positive charged amino acids in the N-
terminal addition.

Analogously, random mutagenesis of the N-terminal addition RPRPRPRP added to the H.
5 lanuginosa lipase variant El*, D57G, N94K, D96L, L97M, Q249R plus other variants were
performed. The nucleotide doping scheme of the RPRPRPRP codons was as follows:

Oligo 2: 5'-GTC TCT GCG TGG ACG GCC TTG G CG GCG CC A CCT CCA 67(T/A)
66(T/A) 575 66CT/A) 67(T/A) 66(T/A) 575 66(T/A) (6/7)(7/8)(C/G) 57(C/G) C57
10 (5/7)5(C/G) CTG TTT AAC CAG TTC AAT CTC-3' (93-mer) (SEQ ID NO 82)
Flask 5: 80% A; 6,66% C; 6,66% G og 6,66 % T.
Flask 6: 80% C; 6,66% A; 6,66% G og 6,66 % T.
FlasK7: 80% G; 6,66% A; 6,66% C og 6,66 % T.
Flask 8 : 80% T; 6,66% A; 6,66% C og 6,66 % G.

APPP is added in the N-terminal of the randomly mutagenized RPRPRPRP and prior to the
signal peptide in order to protect against proteolytic degradation of the N-terminal addition.
This may not be required. El was deleted in order to remove one negatively charged amino
acid. The amino acids in position 2 to 5 of the mature H. lanuginosa lipase sequence were
20 also mutagenized in order to find improved mutants in this non-structural part of the lipase.
Otherwise the procedure is as stated above for the random mutagenesis of SPIRPRP. The
following N-terminal peptide additions were obtained:

Ala-Pro-Pro-Pro- Arg-Pro-Arg-Leu-Leu-Pro-Ile-Ser( APPPRPRLLPIS) (in addtion to the
deleted El residue this variant carries the additional mutation D5E in its non-structural N-
25 terminal part of the mature enzyme).

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Ala-Pro-Pro-Pro-Thr-Arg-Gln-Arg-Gln-Ser-Pro(APPPTRQRQSP) (in addtion to the de-
leted El residue this variant carries the additional mutations V2L, S3T and D5V in its
non-structural N-terminal part of the mature enzyme).

Ala-Pro-Pn>-Pro-Arg-Thr-Ile-Pro-Arg-Ser-Ser-Pro( APPPRTIPRSSP) (in addtion to the
5 deleted El residue this variant carries the additional mutations V2L, S3R and D5E in its
non-structural N-terminal part of the mature enzyme).

Ala-Pro-Pio-Pro-Arg-P^ (APPPRPRPRPRP) (in addtion to the

deleted El residue this variant carries the additional mutations V2G and DSE in its non-
structural N-terminal part of the mature enzyme).

10 Ah-Pro-Pro-PrchArg-Thr-Arg-Pro-Arg-Pro-Arg (APPPRTRPRPRS) (in addtion to the
deleted El residue this variant carries the additional mutations V2GL, S3T, Q4P and
DSE in its non-structural N-terminal part of the mature enzyme).
Ala-Pro-Pro-Pro~Lys-^ (APPPKASPRQRP) (in addtion to the

deleted El residue this variant carries the additional mutations V2GL, D5Q and L6M in

is its non-structural N-terminal part of the mature enzyme).

EXAMPLE 11

Construction of Ps. cepacia lipase variants comprising peptide additions

A lipase gene from Pseudomonas cepacia SB 10, DSM 3959, described in WO
20 89/01032 (from Novo Nordisk A/S) recently rochs^Rcd 3s Burkholderia cepacia wzs clo-
ned, and temperature-inducible expression of the lipase in Escherichia coli was obtained
by use of the plasmid pAHE2. Strain SJ1503 is £. coli JA221 containing pAHE2.

To construct vectors expressing variant lipases with N-terminal extensions, use
were made of two unique restriction sites present in pAHE2, a unique BstXI site appro-
25 ximately 9 codons into the lipase signal peptide coding sequence, and a unique Mlul site
approximately 7 codons downstream from the processing site, Le. in the beginning of the
sequence for the mature lipase.

PCR primers were designed to allow amplification across this region, with the
primers reading upstream from the Mlul site encompassing sequences encoding the N-
30 terminal extensions. All primers had incorporated EcoRI sites in their extreme 5' ends.
The following sequences were chosen to encode N-terminal extensions:

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1) S P I R P R p

AGC CCG ATC CGC CCG CGC CCG

2) T A I R P R K

5 ACG GCG ATC CGC CCG CGC AAG

3) STRRPRP

TCG ACG CGC CGT CCG CGC CCG

io 4) G P I R P R p

GGC CCG ATC CGC CCG CGC CCG

5) S P I R R

AGC CCG ATC CGC CGG

6) RPRPRPRp

CGC CCG CGT CCC AGG CCG CGT CCG
The following primers were used:

LWN9476 (SEQ ID No. 7)(reading downstream from the BstXI site)-
5 '-CGAATTCGATGCGTTCCAGGGTGGTGGCAGG-3 '

LWN9472 (SEQ ID No. 8)(reading upstream from Mlul, designed to incorporate
25 SPIRPRP): v

5'-

CGAATTCACGCGTCGCCGCGTAGCCAGCGGCCGGGCGCGGGCGGATCGGGC
TGGGCGCGGTGGCCGCC ATTGCC-3 *

30 LWN9473 (SEQ ID No. 9)(reading upstream from Mlul, designed to incorporate
TAIRPRX):
5-

GAATTCACGCGTCGCCGCGTAGCCAGCGGCCTTGCGCGGGCGGATCGCCGT
GGGCGCGGTGGCCGCCATTGCC-3 '

LWN9471 (SEQ ID No. 10)(reading upstream from Mlul, designed to incorporate
STRRPRP):

5*-

CGAATTCACGCGTCGCCGCGTAGCCAGCGGCCGGGCGCGGACGGCGCGTCG
40 AGGGCGCGGTGGCCGCCATTGCC-3 '

LWN9474 (SEQ ID No. ll)(reading upstream from Mlul, designed to incorporate

GPffiPRP):

5'-

45 CGAATTCACGCGTCGCCGCGTAGCCAGCGGCCGGGCGCGGGCGGATCGGGC
CGGGCGCGGTGGCCGCC ATTGCC-3 '

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LWN9475 (SEQ ID No. 12)(reading upstream from Mlul, designed to incorporate
SPIRR):

5'-CGAATTCACGCGTCGCCGCGTAGCCAGCGGCCCGGCGGATCGGGCT-
GGGCGCGGTGGCCGCCATTGCC-3 '

LWN9470 (SEQ ID No. 13)(reading upstream from Mlul, designed to incorporate

RPRPRPRP):

5 1 -

CGAATTCACGCGTCGCCGCGTAGCCAGCGGCCGGACGCGGCCTGGGACGCG
10 GGCGGGGCGCGGTGGCCGCC ATTGCC-3 '

For PCR amplifications, primer LWN9476 was used in combination with each of primers
LWN9470-LWN9475, with pAHE2 as template. Annealing temperature was 70°C, and
reactions were performed in the presence of 2% DMSO; otherwise using standard condi-
15 tions and Taq™ polymerase.

Amplified fragments were purified from a 2% agarose gel, digested with BstXI
and Mlul, ligated to the 7. 1 kb BstXI-MluI fragment obtained from pAHE2, and the li-
gation mixture used to transform, by electroporation, & coli SJ6 to ampicillin resistance.
Transformants were plated on LB plates with ampicillin (200 mg/ml) at 30°C.

20 By repiica plating colonies were transferred to lipase screening plates (containing,

pr. litre of agar, 20 ml of Sigma Lipase Substrate (catalogue no. 800-1)) and 4 ml of a
1 % Brilliant Green (Merck, art No. 1.01310) solution), which were incubated at 42°C.
Eventually, green halos, indicating lipase activity, developed around several colonies
from each transformation mixture.

25 Lipase positive colonies were re-isolated, plasmids extracted, and the BstXI-MluI

region DNA sequenced. The following strains were kept:

SJ3606 (SJ6/pSJ3606); contains the SPIRPRP encoding addition, and has also the second
codon in the native, mature enzyme changed from alanine to valine.
SJ3608 (SJ6/pSJ3608); contains a SPRP encoding addition (DNA sequence of insert TCT
30 CCG CGC CCG. Obtained as a variant in attempts to produce a STRRPRP encoding ad-
dition.

SJ3708 (SJ6/pSJ3708); contains the SPIRR encoding addition.
SJ3717 (SJ6/pSJ3717); contains the SPIRPRP encoding addition.
SJ3718 (SJ6VpSJ3718); contains the SPIRPRP encoding addition.

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SJ3719 (SJ6/pSJ3719); contains the TAIRPRK encoding addition.
SJ3720 (SJ6/pSJ3720); contains the STRRPRP encoding addition.
SJ3721 (SJ6/pSJ3721); contains the GPIRPRP encoding addition.

EXAMPLE 12

Shake Flask Fermentatio n of Ps. cepacia Lipase variant

Cultures provide in Example 11 were grown on TY-ampicillin plates (pH 7) and
used to inoculate shake flasks containing 100 ml double concentrated TY-medium with
ampicillin (100 mg/ml) pH 7. The inoculum was checked for lipase productivity (as de-
scribed in the Materials and Methods section) by streaking on indicator plates: all cells
were found to be lipase positive (plates were incubated at 30°C for 2 days, then transfer-
red to 40°C for 1 day).

The shake flasks were incubated shaking at 275 rpm at 30°C for 6 hours until the cul-
tures reached optical densities (578 nm) of 2.8 to 5.3. The cultures were then transferred
to 40°C for another 17 hours.

Check of li pase production in a Ps. cepacia culture

The culture was harvested, centrifuged (20 minutes at 9000 rpm), the supernatant di-
scarded and the pellet re-suspended in NaCl (0.5 ml 0.9% NaCl) and sonicated (2 minu-
tes non-stop, on ice). The sonicated pellet was used to measure Lipase units (LU) using
the titration method with tributyrate as substrate at pH 7.0.
All 8 strains except 1 (SJ3720) showed lipase activity as indicated in the table below.

Strain

time(hs)

OD=578

AmpR:
cell*

OOBGAmp
:cell#

LU/mln

SJ1503wt

t0=0h

0.010

tl=6hs

2.89

t2 = 17hs

7.45

230.5

SJ3606

t0=0h

0.006

tl =6hs

5.24

t2=17hs

9.15

244.45

SJ3608

t0=0h

0.015

tl =6hs

4.40

t2=17hs

9.2

298.6

SJ3708

t0=0h

0.028

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tl=6hs

4.69

t2=17hs

11.05

142.2

SJ3717

t0=0h

0.007

tl=6hs

4.03

t2=17hs

11.2

163.8

SJ3719

t0=0h

0.001

tl=6hs

4.49

t2=17hs

11.7

33.55

SJ3720

t0=0h

0.004

tl=6hs

3.70

t2=17hs

10.5

SJ3721

t0=0h

0.016

tl=6hs

4.20

t2=17hs

11.35

125.75

EXAMPLE 13

5 Characterization of Ps. cepacia Lipase variants

The lipases produced from the strains described In Example 1 1 were characterized
with respect to activity in the presence of detergent, using the PCS plate screening assay
One set of samples was prepared from strains SJ1503, SJ3606 and SJ3608, which had
been propagated as described above, cells harvested, and lysed by sonication to libefate
10 the lipase. 15 ml of samples, containing around 230 LU/ml, were applied in wells in
screening plates either without detergent, or containing 1.5 and 3.5 grams/litre of deter-
gent, respectively. Plates were incubated at 37°C overnight, and the diameter of the green
zone formed around the wells measured. The following results were obtained:

STRAIN

15 SJ1503 SJ3606 SJ3608

DETERGENT

None 17 mm 15 mm 16 mm

1.5 gram/1 7 mm 13 mm 10 mm

3.5 gram/1 0 mm 8 mm 6 mm

Green zones were not observed at higher detergent concentrations.

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Another set of samples were prepared by plating of the strains SJ1503, SJ3708,
and SJ3717-SJ3721 on cellulose acetate filters (each filter containing all 7 strains), which
were placed on LB plates with ampicillin (200 mg/ml) at 37°C overnight, these plates
with filters then incubated at 42°C for 5 hours, after which the filters were transferred
5 (colony side up) to screening plates which were incubated overnight at 37°C.

Pronounced green zones developed under all colonies on the plate without deter-
gent; SJ3720 produced a significantly smaller zone then the rest, most likely due to redu-
ced expression of the lipase.

Green zones were also observed under all colonies on the plate containing 1.5
10 gram/1 of detergent. However, the zone produced from SJ1503, producing the native,
unmodified lipase, was significantly reduced as compared to the zones produced from the
other strains.

On the plate containing 3.5 grams/litre detergent, no green coloration developed
from SJ1503, whereas a greenish stain was still discernible from some strains expressing
15 modified B. cepacia lipases, in particular SJ3717, SJ3718 and SJ3721.

Thus, modification of the B. cepacia lipase gene to encode N-terminal additions
to the native, mature lipase, as those described above, allow the production of lipases
which in the presence of detergent has an improved activity as compared to the native li-
pase.

EXAMPLE 14

Fermentation of 511 SOB and SJ3717 in 10 litre tanks

The method described for shake flask was used for the fermentation in 10 litre
scale. The medium used was Bacto Tryptone 400g, Bacto Yeast extract 200 g, Glucose x
25 2 H 2 0 50Qg, Ampicillin 1 g, Pluronic 1 ml. The pH was kept constant at pH 7.1; the
temperature was 30°C for 7 hours then adjusted to 40°C. Cells were harvested after 16
hours by centrifugation and the cells were opened using a high pressure homogenizer
(800 bar).

30 Purification of B.cqygcia expressed in Exoli

Kcoli cells from 10 liter fermentation broth from SJ1503 and SJ3717 were cen-
trifuged and the supernatant was discarded. Cells were opened using rannie homogenizer

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under pressure 800 bar. Homogenized cells were centrifuged at 350 x g for 60 minutes.
Cell supernatant was decanted.

1. Salt precipitation

5 Activity containing supernatant was precipitated with addition of solid ammonium sulp-
hate to saturation of 35% at room temperature. Precipitation was allowed for 2 hour at
room temperature and centrifuged at 350x g for 1 hour. Supernatant was decanted and
discarded. Precipitate containing activity was dissolved in 30% ethanol to avoid hydrop-
hobic biding of the lipase activity to insoluble material.

io To get rid of insoluble material from the 30 % ethanol dissolved material , the

solution was centrifuged. The lipase activity was recovered as supernatant and insoluble
material was discarded. The supernatant containing activity was concentrated and dialy-
zed against 25 mM Tris-acetate pH 8, by ultra-filtration using Amicon membrane with
cut-off of 10 kDa. The concentrated sample was then diluted five fold in order to reduce

15 any leftover ethanol in the supernatant containing activity.

2. Hydrophobic chromatography

The above sample containing activity was adjusted to 0.8 M ammonium acetate by adding
solid ammonium acetate. 50 ml Toyopearl Butyl column (Tosho Hass, Japan) was packed

20 and equilibrated with 0.8 M ammonium acetate. The samples from above step containing
lipase activity was then adjusted to 0.8 M ammonium acetate and applied on the Toyope-
arl Butyl column. All die activity binds to the matrix. Unbound material was washed with
0.8 M ammonium acetate till Uv absorbence of the effluent was under 0.05 at 280 nm.
Bound activity was eluted with 25 mM Tris acetate buffer containing 50 % ethanol.

25 Fractions containing lipase activity were pooled and dialyzed against 25 mM Tris acetate
buffer pH 8.5.

3. Anion exchange chromatography

50 ml Column was packed with anion exchanger Highperformance Q-sepharose
30 (Pharmacia). The column was washed and equilibrated with 25 mM Tris acetate buffer
pH 8.5 The dialyzed sample was then applied on the column. Unbound activity was

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washed out by using the Tris buffer. Bound activity was eluted with a linear salt gradient
from 0 to 0.5 M NaCl in the Tris buffer pH 8. Flow rate was 2 ml/min and total volume
of the buffer used for elution was 10 column volumes. Fractions containing lipase acti-
vity were pooled and tested for performance in a PCS plate assay.

More specifically, 3 LU of each of the recovered modified lipases were added
into holes of a PCS plate (cf. Example 15 hereinafter) and incubated overnight at 37°C.
After 18 hours the following results were obtained:

STRAIN

io SJ1503 SJ3717

DETERGENT

None 17 mm 13 mm

0.5 gram/1 6 mm 10 mm

L0 gram/1 4 mm 7 mm

Thus, it can be seen that the presence of a peptide addition results in a signifantly higher
wash performance being obtained.

EXAMPLE 15

Construction of modified H. ins olens lipolytic enzymes with an N-terminal peptide ad-
dition

The gene encoding the parent lipolytic enzyme was isolated from Humicola insolens
25 DSM 1800 essentially as described in WO 96/13580. Three different peptide additions
were applied to the N-terminus of the mature enzyme using the plasmid pIVI1303 as the
plasmid template.

Construction of pIVI303 (encoding a H. insolens lipolytic enzyme variant which contains
30 a mutation in the region 304-369 base downstream from ATG without changes in amino
acid sequence and removing a possible secondary DNA structure which might otherwise
have hampered the use of the chameleon double stranded kit.)

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The plasmid was constructed using the Chameleon double-stranded, site-directed
mutagenesis kit from Stratagene (cat no. 200509) according to the described protocol.
pIVI296 was used as the plasmid template and primer no 7258 as the selection

primer.

5 7258: 5*p gaa tga ctt ggt tga cgc gtc acc agt cac 3'

(Thus changing the Seal site found in the ampicillin resistance gene and used for cutting
to a MM site).

Primer no 9349 was used as the mutagenic primer:
9349: 5'p gag tec cac ate cga aac ate tgg ata caa gga gta gga gga cct tac gac gec gcg 3*

1. Variant: HILv4s containing the mutation: PPRRPR (instead of PELVAR in the
native H. insolens propeptide)

Construction of pIVI335:

The plasmid was constructed by use of the Chameleon double-stranded, site- di-
rected mutagenesis kit from Stratagene (cat no, 200509) according to the described proto-
col. pIVI303 was used as a plasmid template.

Primer no. 7887 was used as a selection primer:
7887: :5*p-gaa tga ctt ggt tga gta etc acc agt cac 3*

(changing the introduced Mlul site found in the ampicillin resistance gene and used for
cutting to a Seal site).

Primer no 19473 was used as a mutagenic primer:
19473: 5*p ac cat acc ccg gec get cct cct agg cgt cct egg cag ctg gga gec 3

25 2. Variant: HILvls containing the mutation SPPRRP (instead of ELVARQ in the
native H. insolens propeptide)

Construction of pIVD59:

The plasmid was constructed by use of the Chameleon double-stranded, site-directed
30 mutagenesis kit from Stratagene (cat no. 200509) according to the described protocol.

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pIVD03 was used as a plasmid template. Primer no.7887 (cf. above) was used as a se-
lection primer. Primer no 21992 was used as a mutagenic primer:
21992: 5'p ac cat acc ccg gcc get cct age cct ccg egg egg ccg ctg gga gec ate gag aac ggc
3

3. Variant: HILv2s containing the mutation SPPRP (instead of ELVARQ in the na-
tive H. insolens propeptide)
Construction of pIVI360:

The plasmid was constructed using the Chameleon double-stranded, site-directed mutage-
io nesis kit from Stratagene (cat no. 200509) according to the described protocol.
pIVI303 was used as a plasmid template, and primer no.7887 as a selection primer.
The following primer was used as the selection primer:
5*p ac cat acc ccg gcc get cct age cct ccg egg ccg ctg gga gcc ate gag aac ggc 3

is 4. Variant: HILv3s containing the mutation: SPIRK (instead of ELVARQ in the na-
tive H. insolens propeptide)
Construction of pIVD61:

The plasmid was constructed using the Chameleon double-stranded, site-directed
mutagenesis kit from Stratagene (cat no. 200509) according to the described protocol.

20 pIVI303 was used as a plasmid template and primer no 7887 as a selection primer.
Primer no 21994 was used as a mutagenic primer

21994: 5'p ac cat acc ccg gcc get cct age cct ata cgt aag ctg gga gcc ate gag aac ggc 3

5. Construction of an A. oryzae expression vector

pIVI296:

25 pA2L79 is described in Example 2 of WO 96/13580. The plasmid contains the

H.insolens lipolytic enzyme cDNA sequence inserted into the A. oryzae expression plas-
mid pD414. PA2L79 was cut with the restriction enzymes Hindm and Xhol. The frag-
ment containing the lipolytic enzyme encoding cDNA sequence (1088 bp) was purified
from agarose gel. PHD414 was cut with the restriction enzymes Hindm and Xhol and

30 the vector purified form an agarose gel.

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The purified vector fragment (pHD414) and the lipase containing fragment was ligated
thus creating pIVI296.

Each of the above expression vectors were transformed into A. oryzae IFO 4177
by use of the general transformation method disclosed in the Materials and Methods sec-
5 tion above. One transformant of each type was isolated as HILvl-4s, respectively. The
H. insolens transformants were grown for 3 days in shake flaks at 30°C in 500 ml YPM
medium (10 g/L bacto yeast extract,20 g/L bacto peptone, 20 g/L maltose).

Fermentation supernatent was filtered as described for modified H. lanuginosa
lipolytic enzymes.

10 Purification Step 1:- 1 liter of the Fermentation supernatent was adjusted to pH 8 and di-
luted so conductance of the supernatent was under 4 mSi.

Step 1: Batch treatment of the fermentation supernatent on anion exchanger DE-
AE A50.

DEAE-Sephadex AS0 from Pharmacia was washed and equilibrated with 25 mM tris
is acetate buffer pH 8 using Scintered glass funnel with appropriate pore size. Fermentation

supernatent was then applied on the DEAE Sephadex A50 using the scintered glass fun-

nel. The Lipolytic activity from H. insolence did not bind to anion exchanger at pH 8 and

collected as effluent from DEAE Sephadex A50.

Step 2 :- pH of the efflent from DEAE Sephadex was adjusted to 4.5 by adding
20 dilute Acetic acid. Condctance was also adjusted under 4 mSi by adding water.

Cation Exchange chromatography on SP-Sepharose. 50 ml Column was packed with SP

Sepaharose Fast Flow Code no 17-0729-01 Pharmacia. Column was then washed and e-

quilibrated with 25 mM Sodium acetate buffer pH 4.5.

Sample containing Lipolytic activity adjusted to pH 4.5 and the conductance under 4 mSi
25 was then applied on SP-Sepharose column. Unbound material was washed using 25 mM
Sodium acetate buffer pH 4.5. Lipolytic activity bound to the SP-Sepharose was then
eluted with linear salt gradient with 25 mM Acetate buffer pH 4.5 containing 1M Sodium
Chloride. Fractions containing Lipolytic activity and ratio of the UV absorbence at A280
/A260 was higher than 1.8 were pooled. Purity of the sample was checked on SDS-
30 PAGE.

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Verification of N-terminal peptide addition

The N-terminal amino acid sequence of the HILvls lipolytic enzyme was determined
(i.e. the variant in which the last 5 amino acid residues in the propeptide and the first
amino acid residue in the mature enzyme (ELVARQ) have been substituted with
s SPPRRP).
The N-terminal amino acid sequence found was
Arg-Arg-Pro-Leu-GIy-Ala-Ue-

corresponding to the last three amino acid residues in the substituted sequence and the
first four amino acid residues following the substitution.

The N-terminal amino acid sequence of the HILy2s lipolytic enzyme was also determined
(i.e. in the variant in which the last 5 amino acid residues in the propeptide and the first
amino acid residue in the mature enzyme (ELVARQ) have been substituted with SPPRP).
The N-terminal amino acid sequence found was
is Arg-Pro-Leu-Gly-Ala-Ile-Glu-Asn

corresponding to the last two amino acid residues in the substituted sequence and the first
six amino acid residues following the substitution.

EXAMPLE 16

Characterization of modified Humicola insolens Lipolytic enzymes

The modified lipolytic enzymes comprising peptide additions, produced by the
strains HILvls, HILv2s, fflLv3s, respectively, (described in Example 21), and the wild-
type strain HIL, were characterized with respect to lipase activity on PCS-plates contai-
25 ning 0.5 g/1, 1.0 g/1 and 1.5 g/1 PCS-detergent.

25 pi (corresponding to 5 LU) purified modified HILvsl, HILvs2 and HILvs3
lipase, and wild-type HIL lipase were entered into holes made in the PCS-plates by a pi-
pette (4 mm) and incubated for 3 and 6 hours, respectively.
The result of the test in displayed in the tables below:

Variant

0.5 g/1 PCS-

1.0 g/1 PCS-

1.5 g/1 PCS-

detergent

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HEL

(wild-type)

4 mm

4 mm (weak)

0 mm

HILvls

6 mm

5 mm

4 mm (weak)

HILv2s

5 mm

4 mm

0 mm

HILv3s

6 mm

5 mm

Incubation of 3 hours on PCS-pIates containing FY-detergent

Variant

0.5 gfl PCS-
detergent

1.0 g/1 PCS-
detergent

1.5g/lPCS-
detergent

HIL

(wild-type)

4 mm

4 mm (weak)

0 mm

HILvls

7 mm

5 mm

4 mm (weak)

HILv2s

5 mm

5 mm (weak)

4 mm (weak)

HILv3s

6 mm

4 mm (weak)

Incubation for 6 hours on PCS-plates containing PCS-detergent.
5 As can be seen from the tables the modified lipase variants (/.*. produced by HILvls,
HILv2s and HILvs3) generally have a higher lipase activity in the presence of the PCS-
detergent than the wild-type lipase.

10 EXAMPLE 17

Construction of mo dified H. lanuginosa lipolytic enzvmes with a C-terminal extension

C-terminal peptide additions were applied to the H. lanuginosa lipolytic enzyme variant
is HLvl2s containing the N-terminal peptide addition SPIRPRP and the internal mutations
D57G,N94K,D96L,Q249R.

1. Variant HLvl3s (HLvl2s with the C-terminal peptide addition:
270R^71R t 272P t stop)

20 Construction of plasmid pS14-l:

The plasmid was constructed using the Chameleon double-stranded,site directed mutagenesis
kit from Stratagene (cat no. 200509) according to the described protocol.
pIVI245 was used as the plasmid template (The construction of pIVI245 is described in E-
xample 6) and primer no. 7258 as the selection primer.

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7258: 5'p gaa tga ctt ggt tga cgc gtc acc agt cac 3* (Thus changing the Seal site found in the
ampicillin resistance gene and used for cutting to a Mlul site).

Primer no.20694 was used as the mutagenic primer.
5 20694: 5'p-gg gac atg tct teg acg acc gta gcg get ggg teg act c 3.

2. Variant HLvl4s (HLvl2s with the mutation: 270R,271R,stop)
Construction of plasmid pS20-2:

Hie plasmid was constructed using the Chameleon double-stianded, site-directed mutagenesis
kit from Stratagene (cat no. 20Q5O9) according to the described protocol. pIVI24S was used as
the plasmid template and primer no. 7258 as the selection primer.

7258: 5*p gaa tga ctt ggt tga cgc gtc acc agt cac 3' (Thus changing the Seal site found in the
ampicillin resistance gene used for cutting to a Mlul site).
Primer no. 20695 was used as the mutagenic primer .
20695: 5>gg gac atg tct teg gcg gta ggc gcg get ggg teg ac 3*

Production of qjzyyne v^ri^Q

The enzymes were produced in an analogous manner to that described in Example 6 using
20 the plasmid pToC 202 for the cotransformation step and A. oryzae JAL 125 as a host cell.

Verification of the presence of the C-ter minal extension in HLvl2s

A 1 mg sample of HLvl2s containing the C-terminal extension Arg-Arg-Pro
(RRP) was S-carboxamidomethylated using standard procedures before degradation with

25 a lysyl-specific protease. The resulting peptides were separated using reversed phase
HPLC and the collected fractions subjected to matrix assisted laser desorption ionization
time-of-flight mass spectrometry. A fraction containing a peptide with the experimental
mass of 3906.7 Da was found. This mass is within experimental error identical to the
theoretical mass of the C-terminal peptide of HLvl2s containing the RRP extension

30 which is 3906.4 Da.

The amino acid sequence of the peptide in this fraction was determined to be

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Ile-Glu^ly-De-Asp-AIa-T^
Pro^Asp41c-Pro-Ala-His-Leu-^^
Cys-Leu-Arg-Axg-Pro
s which is the correct amino acid sequence of the C-tenninal peptide of HLvl2s and it
contains the C-tenninal extension Arg-Arg-Pro.

C-tenrrinal extension:

Wash result: 5g/l inactivated Ariel Fumr, 20 minutes, 1 cycle TOM, 30°C, 18°dH. The en-

10 zyme was dosed as mg Enzyme Protein per litre wash solution.
HLvl3s: SPIRPR + D57G f N94K,D96L ( Q249R t 270R f 271R f 272P
HLvl4s: SPIRPR + D57G,N94K,D96L,Q249R,270R,271R

Vaziant

<3R (025 mgEP/1)

dR (1 mgEP/1)

HLvl2s

HLV13s

HLV14s

9.5

EXAMPLE 18

15 A part of the N-tenninal extension of HLvlSs (HLvlSs containing the N-tenninal

peptide addrion SPIRPR and the following mutations in the mature part of the H. lanugi-
nosa lipolytic enzyme EP % D57G, N94K, D96L, L97M, Q249R) was cleaved off by pro-
longed incubation with Clostripain (EC 3.4.22.8; Sigma No. C-0888).

The incubation mixture contained: HLvl5s (1 mg/ml) and Clostripain (20 fig/ml)

20 in 25 mM sodium phosphate, pH 7.4 containing 2.5 mM DTT and 1 mM calcium chlo-
ride.

Before incubation with Clostripain 60% of the lipase carried an intact propeptide
(N-tenninal amino acid sequence SPIRPRP), while 10% had lost the first Ser-residue (N-
terminal amino acid sequence PIRPRPV) and 30% the first 5 amino acid residues of the
25 propeptide (N-tcrminal amino acid sequence (RPVSQDL).

Following incubation for 62 h at ambient temperature (resulting in HLvl5s-C) 60% of
the lipase bad lost the first 4 amino acid residues of the propeptide (resulting in the fol-

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lowing peptide extension PRFVSQ), 20% were without 5 amino acid residues (thus hav-
ing the peptide extension RPVSQD) while the remaining 20% had lost 6 amino acid resi-
dues (thus having the peptide extension PVSQDL).

The propeptide processing was determined using N-tcrminal amino acid sequence
5 determination and it should be noted that the percentages given are approximate values.

Variant

Peptide addition

HLvl5s

60 % SPIRPRPVSQD
10 % PKPRPVSQD
30 % RPVSQD

D57G. N94K, D96L, L97M,
Q249R

HM5s-C

60 % PRPVSQ
20 % RPVSQ
20 % PVSQDL

D57G, N94K, D96L, L97M,
Q249R

l0 One evele wash performance with a modified lipo lytic enzyme treated with clostripain

The one cycle wash performance test (described above in Materials and Methods
section above) was performed with H. lanuginosa lipase variant HLvl5s treated with
clostripain. Wash test was made both with the clostripain treated sample and the non
15 clostripain treated variant. The wash test was carried out in 5 g/1 enzyme inactivated Ariel
Futur (Procter and Gamble). Lard stained swatches were washed for 20 minutes at 30°C.
The tests were performed at lipase concentrations of 0, 5000 LU/1 and 12500 LU/1.

The detegent was dissolved in approx. 18°dH (German Hardness) water. The pH
of the wash liquor was about 10.3. Seven swatches were washed in 1000 ml wash liquor.
20 Subsequent to the washing, ihe swatches were flushed in running up water for 15 minutes
and then air-dried overnight at room temperature.

Evaluation: The reflectance of the swatches was measured at 460 nm, and the
lipase performance (_R) calculated as:

* 25 _R = delta Reflectance =

The mutations of the lipases and the additions arc described above.

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The R, are shown in the table below.

Variant

+/-
treatment w.
clostripain

low dosage

high dosage

HLvl5s

clostripain
treatment

5000 LUA

12500 LU/1

HLvl5s-C

clostripain
treatment

5000 LU/I

12500 LU/1

The results show that the presence of an intact peptide addition leads to the best wash per-
5 fonnance. A reduced (but not entirely removed) peptide addition provides an improved
wash performance, especially when positively charged amino acid residues are present in
the addition.

EXAMPLE 19

10 Modified K lanuginosa lipolytic enzvroe gfmtaminff an cysteine bridee (HLvl6s)

The modified H. lanuginosa lipolytic enzyme HLvl*6s conrains the following mutations:
N94K t D96L, E239C and Q249R and the peptide addition SCERR-
The parent enzyme HLvl6 contains the following mutations: N94K, D96L, Q249R.
15 HLvl6s was constructed as follows:

1. Construction of N94K, D96L mutations in the wildtype B. lanuginosa lipolytic enzyme
Construction of pIVI290:

The plasmid was constructed using the Chamelon double stranded, site-directed mutagenesis
20 kit from Stratagene according to the described protocol using the pAHL (cf Fig. 6 of WO

92/05249) as the plasmid template and primers no 7258 and 7770 as the selection primers.

7258: 5'p gaa tga ctt ggt tga cgc gtc acc agt cac 3' (Thus c h a nging the Seal site found in

the ampicillin resistans gene to a MhJ site)(ScaI has been used for cutting).

7770: Sequence: 5'p tct age cca gaa tac tgg ate aaa tc 3 (Changes' the Seal site found in the
25 wild type H. lanuginosa lipase gene).

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Primer no. 8932 was used as the mutagenic primer.

8932: 5'pgaac tgg ata gga aat ttg aag ttc ctg ttg aaa gaa ata aat gac 3' (Introducing
N94K,D96L)

5 2. Construction of HLvl6s (SCKR, N94K.D96L, E239C, Q249R)
Construction of pIVD19:

The plasmid was constructed using the Chameleon double-stranded,site directed mutagene-
sis kit from Stratagene (cat no. 200509) according to the described protocol using pIVI290
as the plasmid template and primer no 7887 as the selection primer.

10 7887: 5'p-gaatgactt ggt tgagta etc accagicac3 x (changing the introduced Mlul site found
in the ampicillin resistans gene to a Seal site)(MluI has been used for cutting)
Primers no 8829, 9639 and 9646 were used as mutagenic primers
8829; 5 f p-ggc ggc aat aac egg ccg aac art ccg gat ate cc 3' (Introducing Q249R)
9639: 5'p-at ate gtg aag ata tgc ggc atl gat gec acc 3 f (Introducing E239Q

15 9646: 5'p-cg gec ttg get age tgt att cgt cga gag gtc 3* (Modifying the propeptide from
SPERRto SCIKR)

Production of enzymes HLvl6s and HLvl6

The enzymes were produced in an analogous manner to that described in Example 6
20 using A oryviB JAL 125 as a host cell. Subsequently, the one cycle wash performance of
the enzymes were tested (using 5 g/1 of inactivated Ariel Future as detergent and an enzy-
mew dosage of 0.25 mg enzyme protcin/1 and 1.0 mg enzyme protein/1, respectively.

The following results were obtained:

dR (0.25 mg EP/1) dR (1.0 mg EP/1)

HLvl6s 3 7

HLvl6 1 2

30 It is seen that a significantly improved washing performance is obtained for HLvl6s contai-
ning a cystein bridge between the peptide addition and the mature part of the enzyme.

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SEQUENCE LISTING

5 (1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Novo Noidisk A/S

(B) STREET: Novo Alle
10 (Q CITY: Bagsvaerd

(E) COUNTRY: Denmark

(F) POSTAL CODE (ZIP): DK-2880

(G) TELEPHONE: +45 4444 8888

(H) TELEFAX: +45 4449 3256

15 (ii) TITLE OF INVENTION: A modified enzyme with lipolytic activity
fiii) NUMBER OF SEQUENCES: 92
(iv) (iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

20 (Q OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30B (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(Q STRANDEDNESS: single
30 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "synthetic DNA"
(3d) SEQUENCE DESCRIPTION: SEQ ID No. 1:

35 GAATGACTTG GTTGACGCGT CACCAGTCAC 30

(2) INFORMATION FOR SEQ ID No. 2:

40 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(Q STRANDEDNESS: single
(D) TOPOLOGY: linear
45 (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc « "Synthetic DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

TCTAGCCCAG AATACTCGAT CAAATC 26

(2) INFORMATION FOR SEQ ID NO: 3:

0 SEQUENCE CHARACTERISTICS:
55 (A) LENGTH: 55 base pairs

(B) TYPE: nucleic acid

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(Q STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Primer 1"
5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

AACAGATCTT GCGAGACCTC TCTACGTATA GGGCTAGCG A
GCGCGGCGCT GATCG

(2) INFORMATION FOR SEQ ID NO: 4:

0 SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
15 (B) TYPE: nucleic arid

(Q STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) M OLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Primer 2"
20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GTTCTGTCGA ATTGTGAGCG G 21

25 (2) INFORMATION FOR SEQ ID NO: 5:

0 SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 base pairs

(B) TYPE: nucleic arid

30 (Q STRANDEDNESS: single

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE; other nucleic arid

(A) DESCRIPTION: /desc = "Oligo 8479"
(xi*) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

GCGTGGACGG CCTTGGCTAG CCCTATTCGT CCTCGACCGG TCTCGCAGGA

TCTO 54

40 (2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: micfeic arid

45 (Q STRANDEDNESS: single

(D) TOPOLOGY: linear
fri) M OLECULE TYPE: other nucleic arid

(A) DESCRIPTION: /desc « "Primer 3"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

AGAAATCGGG TATCCTTTCA G 21

(2) INFORMATION FOR SEQ ID NO: 7:

0 SEQUENCE CHARACTERISTICS:

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(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(Q STRANDEDNESS: single
(D) TOPOLOGY: linear
5 (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer LWN9476"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

CGAATTCGAT GCGTTCCAGG GTGGTGGCAG G
(2) INFORMATION FOR SEQ ID NO: 8:

15 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 74 base pairs

(B) TYPE: nucleic acid

(Q STRANDEDNESS: single
(D) TOPOLOGY: linear
20 (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer LWN9472"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

25 OGAATTCACG CGTCGCCGCG TAGCCAGCGG CCGGGCGCGG GCGGATCGGG
CTGGGCGCGG TGGCCGCCAT TGCC

(2) INFORMATION FOR SEQ ID NO: 9:

(0 SEQUENCE CHARACTERISTICS:

(A) LENGTH: 74 base pairs

(B) TYPE: nucleic acid

(Q STRANDEDNESS: single
35 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer LWN9473"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO 9:

40 CGAATTCACG CGTCGCCGCG TAGCCAGCGG CCTTGCGCGG GCGGATCGCC
GTGGGCGCGG TGGCCGCCAT TGCC 74

(2) INFORMATION FOR SEQ ID NO: l<h

(0 SEQUENCE CHARACTERISTICS:

(A) LENGTH: 74 base pairs

(B) TYPE: nucleic acid

(Q STRANDEDNESS: single
50 (D) TOPOLOGY: linear

(ii) M OLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer LWN9471 "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO. l(h

55 CGAATTCACG CGTCGCCGCG TAGCCAGCGG CCGGGCGCGG ACGGCGCGTC
GAGGGCGCGG TGGCCGCCAT TGCC 74

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(2) INFORMATION FOR SEQ ID NO: 11:

5 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 74 base pairs

(B) TYPE: nucleic acid

(Q STRANDEDNESS: single
(D) TOPOLOGY: linear
10 (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer LWN9474"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

CGAATTCACG CGTCGCCGCG TAGCCAGCGG CCGGGCGCGG GCGGATCGGG
15 CCGGGCGCGG TGGCCGCCAT TGCC 74

(2) INFORMATION FOR SEQ ID NO: 12:

20 (1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 68 base pairs

(B) TYPE: nucleic acid

(Q STRANDEDNESS: single
(D) TOPOLOGY: linear
25 (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer LWN9475"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

CGAATTCACG CGTCGCCGCG TAGCCAGCGG CCCGGCGGAT CGGGCTGGGC GCGGTGGCCG
30 CCATTGCC 68

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:
35 (A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

40 (A) DESCRIPTION: /desc = "Primer LWN9470"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

CGAATTCACG CGTCGCCGCG TAGCCAGCGG CCGGACGCGG CCTGGGACGC
GGGCGGGGCG CGGTGGCCGC CATTGCC 77

(2) INFORMATION FOR SEQ ID NO: 14:

fi) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 base pairs
50 (B) TYPE: nucleic acid

(Q STRANDEDNESS: single
(D) TOPOLOGY: linear
Co) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Primer 4"
55 (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 14:

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GCTCCTCATG GTGGATCCCC AGTTGTGTAT ATAGAGGATT
GAGGAAGGAA GAGAAGTGTG GATAGAGGTA AATTGAGTTG

GAAACTCCAA GCATGGCATC CTTGC 105
5 (2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 876 base pairs

(B) TYPE: nucleic acid

10 (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:

(B) STRAIN: Humicola lanuginosa DSM 4109
15 (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1 . . 876
(ix) FEATURE:

(A) NAME/KEY: sigj>eptide
20 (B) LOCATION: 1.. 66

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

ATG AGG AGC TCC CTT GTG CTG TTC TIT GTC TCT GCG TGG ACG GCC TTG 48
Met Arg Ser Ser Leu Val Leu Phe Pbe Val Ser Ala Trp Thr Ala Leu
25 1 5 10 15

GCC AGT CCT ATT CGT CGA GAG GTC TCG CAG GAT CTG TTT AAC CAG TTC 96
Ala Ser Pro lie Arg Arg Glu Val Ser Gin Asp Leu Pbe Asn Gin Phe
20 25 30

AAT CTC TTT GCA CAG TAT TCT GCA GCC GCA TAC TGC GGA AAA AAC AAT 144
Asn Leu Phe Ala Gin Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn
35 40 45

35 GAT GCC CCA GCT GGT ACA AAC ATT ACG TGC ACG GGA AAT GCC TGC CCC 192
Asp Ala Pro Ala Gly Thr Asn He Thr Cys Thr Gly Asn Ala Cys Pro
50 55 60

GAG GTA GAG AAG GCG GAT GCA ACG TIT CTC TAC TCG TTT GAA GAC TCT 240
40 Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
65 70 75 80

GGA GTG GGC GAT GTC ACC GGC TTC CTT GCT CTC GAC AAC ACG AAC AAA 288
Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys
45 85 90 95

TTG ATC GTC CTC TCT TTC CGT GGC TCT CGT TCC ATA GAG AAC TGG ATC 336
Leu Be Val Leu Ser Phe Arg Gly Ser Arg Ser lie Glu Asn Trp He
100 105 110

GGG AAT CTT AAC TTC GAC TTG AAA GAA ATA AAT GAC ATT TGC TCC GGC 384
Gly Asn Leu Asn Phe Asp Leu Lys Glu Be Asn Asp He Cys Ser Gly
115 120 125

55 TGC AGG GGA CAT GAC GGC TTC ACT TCG TCC TGG AGG TCT GTA GCC GAT 432
Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp

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130 135 140

ACG TTA AGG CAG AAG GTG GAG GAT GCT GTG AGG GAG CAT CCC GAC TAT 480
Thr Leu Arg Gin Lys Val Glu Asp Ala Val Arg GIu His Pro Asp Tyr
5 145 150 155 160

CGC GTG GTG TIT ACC GGA CAT AGC TTG GGT GGT GCA TTG GCA ACT GTT 528
Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val
165 170 175

GCC GGA GCA GAC CTG CGT GGA AAT GGG TAT GAT ATC GAC GTG TTT TCA 576
Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp He Asp Val Phe Ser
180 185 190

15 TAT GGC GCC CCC CG A GTC GGA AAC AGG GCT TTT GCA GAA TTC CTG ACC 624
Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Pbe Ala Glu Phe Leu Thr
195 200 205

GTA CAG ACC GGC GGA ACA CTC TAC CGC ATT ACC CAC ACC AAT GAT ATT 672
20 Val Gin Thr Gly Gly Thr Leu Tyr Arg He Thr His Thr Asn Asp He
210 215 220

GTC CCT AGA CTC CCG CCG CGC GAA TTC GGT TAC AGC CAT TCT AGC CCA 720
Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro
25 225 230 235 240

GAG TAC TGG ATC AAA TCT GGA ACC CTT GTC CCC GTC ACC CGA AAC GAT 768
Glu Tyr Trp He Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp
245 250 255

ATC GTG AAG ATA GAA GGC ATC GAT GCC ACC GGC GGC AAT AAC CAG CCT 816
He Val Lys He Glu Gly He Asp Ala Thr Gly Gly Asn Asn Gin Pro
260 265 270

35 AAC ATT CCG GAT ATC CCT GCG CAC CTA TGG TAC TTC GGG TTA ATT GGG 864
Asn He Pro Asp He Pro Ala His Leu Trp Tyr Pbe Gly Leu He Gly
275 280 285

ACA TGT CTT TAG 876
40 Thr Cys Leu *
290

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 292 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

50 (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Met Arg Ser Ser Leu Val Leu Phe Phe VaJ Ser Ala Trp Thr Ala Leu
1 5 10 15

55 Ala Ser Pro He Arg Arg Glu Val Ser Gin Asp Leu Phe Asn Gin Pbe
20 25 30

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Asn Leu Phe Ala Gin Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn
35 40 45

5 Asp Ala Pro Ala Gly Thr Asn He Thr Cys Thr Gly Asn Ala Cys Pro
50 55 60

Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
65 70 75 80

Gly Val Gly Asp Val Thr Gly Phe Leu AJa Leu Asp Asn Thr Asn Lys
85 90 95

Leu lie Val Leu Ser Phe Arg Gly Ser Arg Ser He Glu Asn Trp lie
15 100 105 110

Gly Asn Leu Asn Phe Asp Leu Lys Glu He Asn Asp He Cys Ser Gly
115 120 125

20 Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp ArgSerVal AlaAsp
130 135 140

Thr Leu Arg Gin Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr
145 150 155 160

Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val
165 170 175

Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp He Asp Val Phe Ser
30 180 185 190

Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr
195 200 205

35 Val Gin Thr Gly Gly Thr Leu Tyr Arg He Thr His Thr Asn Asp He
210 215 220

Val Pro Arg Leu Pro Pro Arg Glu Pbe Gly Tyr Ser His Ser Ser Pro
225 230 235 240

Glu Tyr Trp He Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp
245 250 255

He Val Lys He Glu Gly He Asp Ala Thr Gly Gly Asn Asn Gin Pro
45 260 265 270

Asn He Pro Asp He Pro Ala His Leu Trp Tyr Phe Gly Leu lie Gly
275 280 285

50 Thr Cys Leu *
290

C2) INFORMATION FOR SEQ ID NO: 17:

55 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids

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(B) TYPE: amino acid

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Arg-Pro-Val-Ser-Gln-Asp
5

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

Ser-Pro-Be-Arg-Met
5

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
25 (B) TYPE: amino acid

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

30 Ser-Pro-De-Arg-AIa-Arg

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Ser-Pro-Ile-Arg-Pro-Arg
5

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

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Ser-PiD-Be-Arg-Ghi-Arg
5

5 (2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid
10 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

Ser-Pro-De-Aig-Lys
15 5

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:
20 (A) LENGTH: 5 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

Ser-Pro-Ile-Lys-Lys
5

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

35 (ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

Ser-Pro-IIe-Arg-Arg-Pro
5

40 (2) INFORMATION FOR SEQ ID NO: 25:

(0 SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid
45 (D) TOPOLOGY: linear

(u) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

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Ser-Pro-Pro-Arg-Arg
5

(2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

10 fii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

Ser-Pro-Iso-Pro-Arg
5

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids
20 (B) TYPE: amino acid

(D) TOPOLOGY: linear
(u) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:

25 Ser-Pro-Arg-Pro-Arg
5

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:
30 (A) LENGTH: 4 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

Ser-Pro-De-Arg

(2) INFORMATION FOR SEQ ID NO: 29:

40 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(u) MOLECULE TYPE: peptide addition
45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

Ser-Pro-De-Arg-Arg
5

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(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:
5 (A) LENGTH: 4 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

SCT-Cys-I}e-Arg-Arg
5

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

Ser-Pro-De-Arg-Pro-Arg-Pro
5

25 (2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid
30 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

Ser-Cys-De-Arg-Pro-Arg-Pro
35 5

(2) INFORMATION FOR SEQ ID NO: 33:

(0 SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
40 (B) TYPE: amino acid

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:

45 Ser*PrcHArg-Arg-Pro-Arg-Thr
5

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(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

5 (ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

Ser-Pio-Phe-Arg-Pro-Lys-Leu
5

(2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
15 (B) TYPE: amino acid

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

20 Ser-Pro-Pro-Arg-Arg-Pro
5

(2) INFORMATION FOR SEQ ID NO: 36:

25 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition
30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

Ser-Pro-De-Arg-Arg-Ghi
5

35 (2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid
40 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

Ser-Pro-Pro-Arg-Pro-Pro
45 5

(2) INFORMATION FOR SEQ ID NO: 38:

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(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

5 (ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

Ser-Pro-Pro-Arg-Pro-Arg
5

10 (2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid
15 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

Ser-Pio-Pro-Trp-Trp-Pro
20 5

(2) INFORMATION FOR SEQ ID NO: 40:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
25 (B) TYPE: amino acid

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

30 Ser-Pio-Pro-Trp-Arg-Pro
5

(2) INFORMATION FOR SEQ ID NO: 41:

35 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition
40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

Ser-Pro-Pio-Arg-Trp-Pro
5

45 (2) INFORMATION FOR SEQ ID NO: 42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

5 Ser-Pro-Pro-Arg-Trp-Pro
5

(2) INFORMATION FOR SEQ ID NO: 43:

10 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition
15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:

Ser-His-Trp-Arg-Arg-Trp
5

20 (2) INFORMATION FOR SEQ ID NO: 44:

0) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid
25 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:

Ser-His-Trp-Arg-Lys
30 5

(2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS:
35 (A) LENGTH: 5 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

Ser-HisTrp-Arg-Arg
5

(2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:

5 Thr-Ala-fle-Arg-Pro-Arg-Lys
5

(2) INFORMATION FOR SEQ ID NO: 47:

10 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition
15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:

Ser-Thr-Arg-Arg-Pro-Arg-Pro
5

20 (2) INFORMATION FOR SEQ ID NO: 48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid
25 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

Gly-Pfo-De-Arg-Pro-Arg-Pro
30 5

(2) INFORMATION FOR SEQ ID NO: 49:

(i) SEQUENCE CHARACTERISTICS:
35 (A) LENGTH: 7 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:

Leu-Pro-Pbe-Arg-Glu-Arg-Pro
5

(2) INFORMATION FOR SEQ ID NO: 50:

(0 SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:

5 Ser-Arg-Ser-Arg-His-Asp-Ala
5

(2) INFORMATION FOR SEQ ID NO: 51:

(i) SEQUENCE CHARACTERISTICS:
10 (A) LENGTH: 7 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

Ile-Pro-De-Arg-Pro-Arg-Arg
5

(2) INFORMATION FOR SEQ ID NO: 52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

25 (ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:

Ser-Hir-Arg-Arg-Pro-Arg-Pro
5

(2) INFORMATION FOR SEQ ID NO: 53:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
35 (B) TYPE: amino acid

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:

40 Thr-Ala-De-Arg-Pro-Arg-Lys
5

(2) INFORMATION FOR SEQ ID NO: 54:

45 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid
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(ii) MOLECULE TYPE: peptide addition

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

Tip-Arg-Trp-Aig-Trp-Arg

(2) INFORMATION FOR SEQ ID NO: 55:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
10 (B) TYPE: amino acid

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

15 Glu-Pro-De-Arg-Arg
5

(2) INFORMATION FOR SEQ ID NO: 56:

20 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition
25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:

Ser-His-Trp-Glu-Ghi
5

30 (2) INFORMATION FOR SEQ ID NO: 57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid
35 (D) TOPOLOGY: linear

fii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:

Arg-Pio-Aig-Pro-Arg-Pro-Arg-Pro
40 5

(2) INFORMATION FOR SEQ ID NO: 58:

fi) SEQUENCE CHARACTERISTICS:
45 (A) LENGTH: 1 1 amino acids

(B) TYPE: amino acid
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:

Ser-Ser-Thr-Arg-Arg-AIa-Ser-Pro-Ile-Lys-Lys
5 10

(2) INFORMATION FOR SEQ ID NO: 59:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1 1 amino acids
10 (B) TYPE: amino acid

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:

15 Ala-Trp-Tip-Pro-Ser-Pro-Ile-Arg-Pro-Arg-Pro
5 10

(2) INFORMATION FOR SEQ ID NO: 60:

20 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition
25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:

Ala-Pro-Pro-Pro-Arg-Pro-Arg-Pio-Ai^-Pro-Arg-Pro
5 10

30 (2) INFORMATION FOR SEQ ID NO: 6 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid
35 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:

Ala-Pro-Pro-Pio-Arg-Thr-Arg-Pro-Arg-Pro-Afg^
40 5 10

(2) INFORMATION FOR SEQ ID NO: 62:

(i) SEQUENCE CHARACTERISTICS:
45 (A) LENGTH: 8 amino acids

(B) TYPE* amino acid
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:

Ser-Pro-Lys-Arg-Lys-Pio-Arg-Pro
5

(2) INFORMATION FOR SEQ ID NO: 63:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
10 (B) TYPE: amino acid

(D) TOPOLOGY: linear
fii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:

15 Ser-GIn-Arg-Ile-Lys<5In-Arg-Ile-Lys
5

(2) INFORMATION FOR SEQ ID NO: 64:

20 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition
25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:

Ser-Pio-Pro-Pro-Arg-Pro-Arg-Pro
5

30 (2) INFORMATION FOR SEQ ID NO: 65:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid
35 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:

Ser-Pro-IIe-Arg-Pro-Ai^Pro-Arg*Pro-Arg
40 5 10

(2) INFORMATION FOR SEQ ID NO: 66:

(i) SEQUENCE CHARACTERISTICS:
45 (A) LENGTH : 9 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:

v Ser-Pro-De-Aig-Lys-Ala-Trp-Trp-Pro
5

(2) INFORMATION FOR SEQ ID NO: 67:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
10 (B) TYPE: amino acid

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:

15 Ala-Pro-Pro-Pro-Lys-Ala-Ser-Pro-Arg-Gln-Arg-Pro
5 10

(2) INFORMATION FOR SEQ ID NO:68:

20 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid
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(ii) MOLECULE TYPE: peptide addition
25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:

Ser-Pro-Ile-Arg-Pro-Arg-Pro-Ser^

5 10 15

30 (2) INFORMATION FOR SEQ ID NO: 69:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

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(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:

Ser-Pro-Pro-Arg-Tip-Pro-Arg-Arg
40 5

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(i) SEQUENCE CHARACTERISTICS:
45 (A) LENGTH: 8 amino acids

(B) TYPE: amino acid
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(ii) MOLECULE TYPE: peptide addition

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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:

Ser-Pro-Pn>-Arg-Trp-Pro-Afg-Trp
5

(2) INFORMATION FOR SEQ ID NO: 71:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids
10 (B) TYPE: amino acid

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:

15 Ser-Pio-Pro-Arg-TTp-Pro-Trp-Arg
5

(2) INFORMATION FOR SEQ ID NO: 72:

fi) SEQUENCE CHARACTERISTICS:
20 (A) LENGTH: 8 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:

Ser-Pro-Prc-Trp-Arg-Pro-Arg-Arg
5

(2) INFORMATION FOR SEQ ID NO: 73:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid
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35 (ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:

SeMW-Pro-Trp-Trp-Pro-Arg-Trp
5

(2) INFORMATION FOR SEQ ID NO: 74:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids *
45 (B) TYPE: amino acid

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:

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Ser-Pro-Pro-Tip-Tq)-PrD-Trp-Arg
5

5 (2) INFORMATION FOR SEQ ID NO: 75:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid
10 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:

Ser-Pio-Pro-Trp-Trp-Pro-Trp-Trp
15 5

(2) INFORMATION FOR SEQ ID NO: 76:

(i) SEQUENCE CHARACTERISTICS:
20 (A) LENGTH: 8 amino acids

(B) TYPE: amino acid
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(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:

Ser-Pro-Pio-Trp-Pn>-Arg-Pro-Arg-Pio
5

(2) INFORMATION FOR SEQ ID NO: 77:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic arid

(Q STRANDEDNESS: single
35 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer 7887*
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:

40 gaatgacttg gttgagtact caccagtcac 30

(2) INFORMATION FOR SEQ ID NO: 78:

45 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 46 base pairs

(B) TYPE: nucleic arid

(Q STRANDEDNESS: single
(D) TOPOLOGY: linear
50 (ii) MOLECULE TYPE: other nucleic arid

(A) DESCRIPTION: /desc = "Primer 8932"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:

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gaactggata ggaaatttga agttcctgtt gaaagaaata aatgac

(2) INFORMATION FOR SEQ ID NO: 79:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer 7258"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:

IS gaatgacttg gttgacgcgt caccagtcac

(2) INFORMATION FOR SEQ ID NO: 80:

(i) SEQUENCE CHARACTERISTICS:
20 (A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

25 (A) DESCRIPTION: /desc « "Primer 7770"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:

tctagcccag aatactggat caaatc

(2) INFORMATION FOR SEQ ID NO: 81:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs

35 (B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer 8479"
40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:

gcgtggacgg ccttggctag ccctattcgt cctcgaccgg tctcgcagga tctg

45 (2) INFORMATION FOR SEQ ID NO: 82:

0 SEQUENCE CHARACTERISTICS:

(A) LENGTH: 57 base pairs

(B) TYPE: nucleic acid

50 (Q STRANDEDNESS: single

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Obgo I"
(xi*) SEQUENCE DESCRIPTION: SEQ ID NO: 82:

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GCGTGG ACGG CCTTGGCC86(T/A) 66( A/T) 58(T/A) 67(T/A) 66(T/A) 575 66(T/A) GAGGTC TCG
CAGGATC TG

(2) INFORMATION FOR SEQ ID NO: 83:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 93 base pairs

(B) TYPE: nucleic acid

(Q STRANDEDNESS: single
10 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = 'Oligo Y

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:

15 GTCTCTGCGT GGACGGCCTT GGCGGCGCCA CCTCCA67(T/A) 66(T/A) 575 66<T/A) 67{T/A)
66(T/A) 575 66(T/A) (6/7)(7/8)(C/G) 57(C/G) C57 (5/7)5(C/G) CTGT TTAACCAGTT CAATCTC

(2) INFORMATION FOR SEQ ID NO: 84:

20 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(Q STRANDEDNESS: single
(D) TOPOLOGY: linear
25 (ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer 7887"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:

gaatgacttg gttgagtact caccagtcac 30

(2) INFORMATION FOR SEQ ID Na 85:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base pairs

35 (B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = 'Primer 19473'
40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:

accatacccc ggccgctcct cctaggcgtc ctcggcagct gggagcc 47

45 (2) INFORMATION FOR SEQ ID NO: 86:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 59 base pairs

(B) TYPE: nucleic acid

50 (Q STRANDEDNESS: single

(D) TOPOLOGY: linear

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fu) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "Primer 21992"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:

5 accatacccc ggccgctcct agccctccgc ggcggccgct gggagccatc gagaacggc 59

(2) INFORMATION FOR SEQ ID NO: 87:

(i) SEQUENCE CHARACTERISTICS:
10 (A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(Q STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

15 (A) DESCRIPTION: /desc = "Primer 21 994"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:

acc at ac cc c ggccgctcct agccctatac gtaagctggg agccatcgag aacggc 56

(2) INFORMATION FOR SEQ ID NO: 88:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 56 base pairs

25 (B) TYPE: nucleic acid

(Q STRANDEDNESS: single
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = 'Primer 9349"
30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:

gagtcccaca tccgaaacat ctggatacaa ggagtaggag gaccttacgac gccgcg 56

35 (2) INFORMATION FOR SEQ ID NO: 89:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid
40 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:

Ser- Ala-Leu- Arg-Pro-Arg-Lys
45 5

(2) INFORMATION FOR SEQ ID NO: 90:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
50 (B) TYPE: amino acid

(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:

SUBSTITUTE SHEET (RULE 26)

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Ala-Pro-Pio-Pio-Arg-Pro-Aig-Leu-Leu-Pro-De-Ser

5 (2) INFORMATION FOR SEQ ID NO: 91 :

(0 SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1 1 amino acids

(B) TYPE: amino acid
10 (D) TOPOLOGY: linear

fii) MOLECULE TYPE: peptide addition

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:

AIa-rVo-Pro-Pro-Th/-Arg<51fi-Arg^ln-Ser>Pro
15 5 10

(2) INFORMATION FOR SEQ ID NO: 92:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide addition
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:

Ala-Pro-Pro-Pro- Arg-Thr-I!e-Pn>Arc-Ser-Ser-Pro
5 10

(2) INFORMATION FOR SEQ ID NO: 93:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 288 amino acids

(B) TYPE: amino acid

(Q STRANDEDNESS: single
35 (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein
(vi) ORIGINAL SOURCE:

(B) STRAIN: Peudomonas sp.

40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:

Phe Gly Ser Ser Asn Tyr Thr Lys Thr Gin Tyr Pro He Val LeuThr
15 10 15

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His Gly Met Leu Gly Phe Asp Ser Leu Leu Gly Val Asp Tyr Trp Tyr
20 25 30

Gly lie Pro Ser Ala Leu Arg Lys Asp Gly Ala Thr Va! Tyr Val Thr
5 35 40 45

Glu Val Ser Gin Leu Asp Thr Ser Glu Ala Arg Gly Glu Gin Leu Leu
50 55 60

10 Thr Gin Val Glu Glu lie Val Ala He Ser Gly Lys Pro Lys Val Asn
65 70 75 80

Leu Phe Gly His Ser His Gly Gly Pro Thr He Arg Tyr Val Ala Ala
85 90 95

Val Arg Pro Asp Leu Val Ala Ser Val Thr Ser He Gly Ala Pro His
100 105 no

Lys Gly Ser Ala Thr Ala Asp Phe He Arg Gin Val Pro Glu Gly Ser
20 115 120 125

Ala Ser Glu Ala He Leu Ala Gly He Val Asn Gly Leu Gly Ala Leu
130 135 140

25 He Asn Phe Leu Ser Gly Ser Ser Ser Asp Thr Pro Gin Asn Ser Leu
145 150 155 160

Gly Thr Leu Glu Ser Leu Asn Ser Glu Gly Ala Ala Arg Phe Asn Ala
165 170 175

Arg Phe Pro Gin Gly Val Pro Thr Ser Ala Cys Gly Glu Gly Asp Tyr
180 185 190

Val Val Asn Gly Val Arg Tyr Tyr Ser Trp Ser Gly Thr Ser Pro Leu
35 195 200 205

Thr Asn Val Leu Asp Pro Ser Asp Leu Leu Leu Gly Ala Thr Ser Leu
210 215 220

40 Thr Phe Gly Phe Glu Ala Asn Asp Gly Leu Val Gly Arg Cys Ser Ser
225 230 235 240

Arg Leu Gly Met Val lie Arg Asp Asn Tyr Arg Met Asn His Leu Asp
245 250 255

Glu Val Asn Gin Thr Phe Gly Leu Thr Ser He Phe Glu Thr Ser Pro
260 265 270

Val Ser Val Tyr Arg Gin Gin Ala Asn Arg Leu Lys Asn Ala Gly Leu
50 275 280 285

SUBSTITUTE SHEET (RULE

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PATENT CLAIMS

1. A modified enzyme with lipolytic activity which as compared to its parent enzyme has
i) a peptide addition in its C-terminus or N-terminus or ii) peptide additions in its C-
terminus and its N-terminus.

2. The modified enzyme according to claim 1, which comprises a peptide addition in its
N-terminus,

3. The modified enzyme according to claim 1 or 2, wherein the peptide addition is selected
so as to increase the affinity of the parent enzyme towards its substrate.

4. The modified lipolytic enzyme according to any of claims 1-3, wherein the peptide ad-
dition is selected so as to confer stability to the modified lipolytic enzyme.

5. The modified lipolytic enzyme according to claim 4, wherein the peptide addition is one
which is capable of forming a covalent binding to the mature part of the parent enzyme.

6. The modified enzyme according to any of claims 1-5, which comprises a cysteine resi-
due in the peptide addition and a cystein residue in the mature part of the parent enzyme
in such a manner that the said cystein residues together forms a cystein bridge.

7. The modified lipolytic enzyme according to claim 6, wherein the cystein residue in the
mature part of the parent enzyme has been inserted or has replaced and amino acid resi-
due of the parent enzyme.

8. The modified lipolytic enzyme according to any of claims 1-7, wherein the peptide ad-
dition is one which has a low susceptibility to proteolytic degradation by proteolytic en-
zymes of a host cell used for expressing the modified lipolytic enzyme.

9. The modified lipolytic enzyme according to claim 8, wherein the peptide addition com-
prises at least one proline residues, such as two or three proline residues.

10. The modified enzyme according to any of claims 1-9, wherein the peptide addition
comprises at least one, such as one, two or three positive or hydrophobic amino acids
residues.

11. The modified enzyme according to any of claims 1-10, wherein the length of the pep-
tide addition is from 1 and 500 amino acids, preferably 1 to 200, more preferably 2 to
100, even more preferably 2 to 50, and most preferably between 1 and 15, such as 1 to
10 or 4 to 10 amino acids.

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12. The modified enzyme according to any of claims 1-11, wherein the peptide addition is
one of the following peptide additions:

Arg (R), or Lys (K), or Leu (L), or He (I), or

Val (V), or Trp (W) or Phe (F), or
5 Arg-Pro (RP), or

Lys-Lys (KK), or

Arg-Lys (RK), or

Lys-Arg (KR), or

Arg-Arg (RR), or
10 Arg- Arg-Pro (RRP), or

Arg-Pro- Val-Ser-Gln-Asp (RPVSQD)

Ser-Pro-De-Arg-Met (SPIRM), or

Ser-Pro-rJe-Arg-Ala-Arg (SPIRAR), or

Ser-Pro-Ue-Arg-Pro-Arg (SPIRPR) or
is Ser-Pro-Ile-Arg-Glu-Arg (SPIRER), or

Ser-Pro-Ile-Arg-Lys (SPIRK), or

Ser-Pro-ne-Lys-Lys (SPKK), or

Ser-Pro-He-Arg-Arg-Pro (SPIRRP), or

Ser-Pro-Pro- Arg-Arg (SPPRR), or
20 Ser-Pro-Iso-Pro-Arg (SPIPR), or

Ser-Pro-Arg-Pro-Arg (SPRPR), or

Ser-Pro-ne-Arg (SPIR), or

Ser-Pro-De-Arg-Arg (SPIRR), or

Ser-Cys-ile- Arg-Arg, (SCIRR), or
25 Ser-Pro-rJe-Arg-Pro-Arg-Pro (SPIRPRP), or

Ser-Cys-De- Arg-Pro- Arg-Pro (SCPIRPRP), or

Ser-Pro-Arg-Arg-Pro-Arg-Thr (SPRRPRT), or

Ser-Pro-Phe-Arg-Pro-Lys-Leu (SPFRPKL), or

Ser-Pro-Pro-Arg-Arg-Pro (SPPRRP), or
30 Ser-Pro-De-Arg-Arg-Glu (SPIRRE), or

Ser-Pro-Pro-Arg-Pro-Pro (SPPRPP), or

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Ser-Pro-Pro-Arg-Pro-Arg (SPPRPR), or

Ser-Pro-Pro-Trp-Trp-Pro (SPPWWP), or

Ser-Pro-Pro-Trp-Arg-Pro (SPPWRP), or

Ser-Pro-Pro-Arg-Trp-Pro (SPPRWP), or
5 Ser-Pro-Pro-Arg-Trp-Pro (SPPRWP), or

Ser-His-Trp-Arg-Arg-Trp (SHWRRW), or

Ser-His-Trp-Arg-Lys (SHWRK), or

Ser-His-Trp-Arg-Arg (SHWRR), or

Thr-Ala-De-Arg-Pro-Arg-Lys (TAIRPRK),
10 Ser-Thr-Arg-Arg-Pro-Arg-Pro (STRRPRP),

Gly-Pio-Ile-Arg-Pro-Arg-Pro (GPIRPRP),

Leu-Pro-Phe-Arg-Glu-Arg-Pro (LPFRQRP),

Ser-Arg-Ser-Arg-His-Asp-Ala (SRSRHNA),

ne-Pro-De-Arg-Pro-Arg-Arg (EPIRPRR),
is Ser-Thr-Arg-Arg-Pro-Arg-Pro (STRRPRP),

Thr-Ala-De-Arg-Pro-Arg-Lys (TAIRPRK),

Trp-Arg-Trp-Arg-Trp-Arg (WRWRWR),

Glu-Pro-De-Arg-Arg (QPIRR),

Ser-His-Trp-Glu-Glu (SHWQQ),
20 Arg-Pro-Aig-Pro-Arg-Pro-Arg-Pro (RPRPRPRP), or

Ser-Ser-Thr-Arg-Arg-Ala-Ser-Pro-De-Lys-Lys (SSTRRASPIKK), or

Ala-Trp-Trp-Pro-Ser-Pro-Ile-Arg-Pro-Arg-Pro (AWWPSPIRPRP), or

Ala-Pro-Pro-Pro-Aig-Pro-Arg-Pro-Arg-Pro-Arg-Pro (APPPRPRPRPRP), or

Ab-Pio-Pro-Pro-Aig-Thr-Arg-Pro-Arg-Pro-Arg-Ser (APPPRTRPRPRS), or
25 Ser-Pro-Lys-Arg-Lys-Pro-Arg-Pro (SPKRKPRP), or

Ser-Gln-Arg-De-Lys-Gln-Arg-Ile-Lys (SQRIKQRK), or

Ser-Pro-Pro-Pro-Arg-Pro-Arg-Pro (SPPPRPRP), or

Ser-Pro-De-Arg-Pro-Arg-Pro-Arg-Pro-Arg SPIRPRPRPR, or

Ser-Pro-ne-Arg-Lys-Ala-Trp-Trp-Pro (SPIRKAWWP), or
30 Ala-Pro-Pro-Pro-Lys-Ala-Ser-Pro-Arg-Gln-Arg-Pro (APPPKASPRQRP), or

Ser-Pro-ne-Arg-Pro-Arg-Pro-Ser-Pro-Ile-Arg-Pro-Arg-Pro-Arg (SPIRPRPSPIRPRP), or

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Ser-Pro-Pro-Aig-TqvPro-Arg-Arg (SPPRWPRR), or
Ser-Pro-Pro-Arg-Trp-Pro-Arg-Trp (SPPRWPRW), or
Ser-Pro-Pro-Arg-Trp-Pro-Trp-Arg (SPPRWPWR), or
Ser-Pro-Pro-Trp-Arg-Pro-Arg-Aig (SPPWRPRR), or
5 Ser-Pro-Pro-Trp-Tip-Pro-ATg-Trp (SPPWWPRW, or
Ser-Pro-Pro-Tip-Tip-Pro-Trp-Arg (SPPWWPWR), or
SCT-Pro-Pro-Tip-Trp-Pro-Tip-Trp (SPPWWPWW), or
Ser-Pro-Pro-Tip-Pro-Arg-pno-Arg-Pro (SPPWPRPRP).

10 13. The modified enzyme according to any of claims 1-12, which, in addition to the pep-
tide addition, comprises a mutation in a non-structural part of the N-terminus and/or C-
terminus of the parent enzyme, in particular a mutation which has resulted in the removal of
at least one negatively charged amino acid residue.

14. The modified enzyme according to claim 13, wherein a negatively charged amino add
15 residue of said non-structural part has been deleted or replaced by a neutral or positively

charged amino acid residue or by a hydrophobic amino acid residue, or wherein a neutral
amino acid residue has been replaced with a positively charged amino acid residue.

15. A modified lipolytic enzyme comprising a peptide addition according to any of claims
1-14, said peptide addition having been applied by

20 a) subjecting a DNA sequence encoding a parent enzyme with a peptide addition to ran-
dom mutagenesis in the part of the DNA sequence encoding the peptide addition and
optionally a non-structural N-terminal or C-terminal part of the mature form of the par-
ent enzyme,

b) expressing the resulting mutated DNA sequence in a suitable host cell so as to produce a
25 modified lipolytic enzyme, and

c) screening for modified lipolytic enzymes resulting from step c) which has an improved
performance as compared to the parent enzyme.

16. The modified lipolytic enzyme according to claim 14, wherein the random mutagenesis
is performed so as to introduce one or more positively charged or hydrophobic amino acid

30 residues into the peptide addition and optionally the non-structural part of the parent en-
zyme.

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17. The modified enzyme according to any of claims 1-16 wherein the enzyme is of mi-
crobial origin.

18. The modified enzyme according to claim 17, wherein the enzyme is of bacterial, yeast
or filamentous fungal origin.

5 19. The modified enzyme according to claim 18, wherein the enzyme is derived from
Hwmcola sp., such from ft lanuginosa or ft insolens.

20. The modified lipolytic enzyme according to claim 19, which is derived from the ft.
lanuginosa strain DSM 4109 and having the amino acid sequence shown in Fig. L

21. The modified enzyme according to claim 20, which further comprises a mutation in a
10 non-structural part of the C-terminus or N-terminus of the parent enzyme, preferably a muta-
tion which has resulted in the removal of a negative charged amino acid residue.

22. The modified enzyme according to claim 21, in which the peptide addition has re-
placed the amino acid residue occupying position 1 in the mature parent enzyme, i.e. the
El.

15 23. The modified enzyme according to claim 18, wherein the enzyme is derived from
Pseudomonas sp., in particular ft. cepacia, or Ps. mendocina or ft. alcaligenes or ft. psext-
doalcaligenes or ft. plantarii or ft. gladioli, or ft. putida, or Ps. aeruginosa, or ft.
Glumae.

24. The modified enzyme according to any of claims 1-23, wherein the parent enzyme is
20 in a mature form.

25. The modified enzyme according to any of claims 1-24, wherein the parent enzyme is a
naturally-occuring enzyme or a variant thereof.

26. The modified enzyme according to any of claims 1-25, wherein the peptide addition is
different from the native pre, pro or preprosequence of the parent enzyme.

25 27. A DNA sequence encoding a modified enzyme exhibiting lipolytic activity according
to any of claims 1-26, provided that the part of the DNA sequence encoding the peptide addi-
tion is different from the DNA sequence which is normally associated with, and encodes the
profbrm or preproform of, the parent enzyme.

28. A recombinant vector or transformation vehicle comprising the DNA sequence ac-

30 cording to claim 27.

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29. The vector according to claims 27 and 28, which is an expression vector further com-
prising DNA sequences permitting expression of the enzyme.

30. A host cell harbouring a DNA sequence according to claim 27 or a vector according
to claim 28 or 29.

5 31. The host cell according to claim 30, which is a microbial cell, such as a filamentous
fungal, a yeast or a bacterial cell.

32. The host cell according to claim 31 , which is a strain of a Aspergillus sp., such as A.
niger, A. oryzae and A. japonicus, or a strain of Fusarium sp., such as a strain of F. ax-
ysporum or F. gramineanan.

10 33. The host cell according to claim 31 , which is a cell of a gram-positive bacterial strain,
e.g. of the genus Bacillus, such as B. sub rilis, B. lidxeniformis, B. lemus, B. brew, B. stea-
rothermophilus, B. alkabphilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus,
B. thwingiensis, or of the genus Streptomyces y . or a cell of a gram-negative bacterial strain,
such as Kcoli y oT of the genus Pseudomonas.

is 34. The host cell according to any of claims 30-33, which has been modified so as to have
a reduced production of one or more proteolytic enzymes compared to the unmodified host
cell, e.g. a host cell which has been made deficient in one or more proteolytic enzymes.

35. A process for preparing a modified enzyme according to any of claims 1-25 compris-
ing

20 a) cultivating a host cell according to any of claims 30-34 under conditions conducive for the
production of the modified enzyme, and
b) recovering and optionally purifying the resulting enzyme.

36. The method according to claim 35, wherein the host cell, the cultivation conditions and/or
recovery conditions being selected so that at least 5% of the produced modified enzyme com-

25 prises the peptide addition encoded by the peptide addition.

37. A method of improving properties, such as increasing the affinity towards a lipid sub-
strate, of a parent lipolytic enzyme, which method comprises applying a peptide addition to
the N-terminus or C-terminus of the parent enzyme in its mature form.

38. The method according to claim 37, wherein the improved properties is an improved wash
30 performance.

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39. The method according to claim 37 or 38, wherein the peptide addition is applied to the
parent enzyme by cultivating a host eel] comprising a DNA sequence encoding the pre, pro or
preproform of the parent lipolytic enzyme, the DNA sequence optionally being present cm a
vector, and recovering the resulting modified lipolytic enzyme, the host cell, cultivation con-

5 ditions and/or recovery conditions being selected so that at the most a partial processing of the
pre, pro or preproform of the parent enzyme has occurred resulting in that at least 5% of the
produced modified enzyme molecules comprises the desired peptide addition, e.g. the entire
prosequence or a substantial part thereof.

40. The method according to claim 37 or 38, wherein the peptide addition is applied to the
to parent enzyme by cultivation of a host cell comprising a DNA sequence according to claim 27

or a vector according to claim 28 or 29, and recovering the resulting modified lipolytic en-
zyme,

the host cell, cultivation conditions and/or recovery conditions being selected so that at least
5% of the produced modified enzyme comprises the peptide addition encoded by the peptide
15 addition.

41. The method according to claim 40, wherein the host cell is of a different origin than
the parent enzyme, e.g. of another genus than the one from which the parent enzyme is de-
rived, or has another posttranslational processing machinery than the source of the parent en-
zyme.

20 42. The method according to claim 41, wherein the parent lipolytic enzyme is derived
from a filamentous fungus or a bacterium and the host cell is a yeast cell.

43. The method according to claim 42, wherein the parent lipolytic enzyme is derived
from a strain of a Hwnicola sp. y such as H. lanuginosa or from a strain of a Pseudomonas sp.

44. The method according to any of claims 39-43, wherein the host cell is a yeast cell,
25 sue* as a strain of Saccharvmyces sp., in particular Saccharomyces cerevisiae, or a strain of

Hanserudasp..

45. The method according to any of claims 39-44, wherein the inherent proteolytic enzyme
producing capability of the host cell used for applying the peptide addition to the parent
lipolytic enzyme has been reduced, e.g. by abolishing the production of one or more prote-

30 olytic enzymes by the host cell.

46. A method according to claim 37, comprising

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a) subjecting a DNA sequence encoding the parent lipolytic enzyme with a peptide addition,
such as a DNA sequence according to any of claims 1-26, to localized random mutagene-
sis in the part of the DNA sequence encoding the peptide addition or a non-structural part
of the C-terminal or N-terminal end of the parent enzyme,
5 b) expressing the mutated DNA sequence obtained in step a) in a host cell, and

c) screening for host cells expressing a mutated lipolytic enzyme which has an improved per-
formance as compared to the parent lipolytic enzyme.

47. The method according to claim 46, wherein the DNA sequence is the gene or cDNA
sequence encoding the parent enzyme in its pro or prepro form.
10 48. The method according to any of claims 37-47, which further involves introducing a
mutation in a non-structural part of the C-terminus or N-terminus of the parent enzyme in its
mature form.

49. The method according to claim 48, wherein the mutation involves deleting or replacing
a negatively charged amino add residue of the non-structural part with a neutral or positively

15 charged amino acid residue or with a hydrophobic amino acid residue, or replacing a neutral
amino acid residue with a positively charged amino acid residue.

50. An enzyme composition comprising a modified enzyme with lipolytic activity accord-
ing to any of claim 1-26.

51. The composition according to claim 50, which further comprise at least one enzyme
20 selected from the group of proteases, ceDulases, peroxidases, cutinases, amylases and/or li-
pases.

52. A detergent composition comprising an enzyme composition according to claim 50 or
51.

53. The composition according to claim 52, which contains between 0.02 and 200 mg of
25 modified lipolytic enzyme protein/g detergent composition.

54. The composition according to any of claims 50 and 52, wherein more than 5%, pref-
erably more than 10%, such as 25%, better 50% especially 75% of the modified lipolytic en-
zyme in the composition has a full length peptide addition.

55. Use of an enzyme composition according to any of claims 50-54, in detergents, such as
30 washing powder or dishwashing compositions.

SUKTT ^Sf£ET(RulE26)

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ATGAGGAGCTCCCTTGTGCTGTTCTTTGTCTCTGCGT^
1 MRSSLVLFFVSAWTALASPI

CGTCGAGAGGTCTCGCAGGATCTGTTTAACCAGTTCAATCTCTTTGCACAGTATO
21 KREVSQDLFNQFNLFAQYSA

GCCG CATACTGCGGAAAAAACAATGATGCCCCAGCTGGTACAAACATTACGTG CACGGGA
41 AAYCGKNNDAPAGTNITCTG

AATGCCTGCCCCGAGGTAGAGAAGGCGGATGCAACGTTTCTCTACTCGTCT
61 NACPEVEKADATFLYSFEDS

GGAGTGGGCGATGTCACCGGCTTCCTTGCTCTCGACAACACGM
81 GVGDVTGFLALDNTNKLIVL

TCTTTCCGTGGCTCTCGTTCCATAGAGAACTGGATCGGGAATCT^
101 SFRGSRSIENWIGNLNFDLK

GAAATAAATGACATTTGCTCCGGCTGCAGGGGACATGACOT
121 EINDICSGCRGHDGFTS SWR

TCTGTAGCCGATACGTTAAGGCAGAAGGTGGAGGATGCTGTGAGGGAGCA
141 SVADTLRQKVEDAVREHPDY

CGCGTGGTGTTTACCGGACATAGCTTGGGTGGTGCATTGGCAACT^
161 RVVFTGHSLGGALATVAGAD

CTGCGTGGAAATGGGTATGATATCGACGTGTTTTCATATGGCGCCCCCCGAGTCGGAAAC
181 LRGNGYDIDVFSYGAPRVGN

AGGGCTTTTGCAGAATTCCTGACCGTACAGACCGGCGGAAC^

201 RAFAEFLTVQTGGTLYRITH

ACCAATGATATTGTCCCTAGACTCCCGCCGCGCGAATTCGGTTAC^
221 TNDIV PRLPPREFGYSHSSP

241 EYWIKSGTLVPVTRNDIVKI

GAAGGCATCGATGCCACCGGCGGCAATAACCAGCCTAACATTCCGGATATCCCTC
261 EGIDATGGNNQPNIPDIPAH

CTATGGTACTTCGGGTTAATTGGGACATGTCTTTAG
281 LWYFGLIGTCL*

Fig. 1

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ATGAAACGCATTTGTGGTTCCCTGCTGTTGCTCGGT^
1 MKRICGSLLLLGLSISAALA

AGCCCTATACGTAGAGAGGTCTCGCAGGATCTGTTTAACCAGTTCAATCTCT^
21SPIRREVSQDLFNQFNLFAQYSA

GCCGCATACTGCGGAAAAAACAATGATGCCCCAGCTGGTACAAAC^
44 AAYCGKNNDAPAGTNITCTG

N A C P E

rCGTTTGAAGACTCT
VEKADATFLYS FEDS

GGAGTGGGCGATGTCACCGGCTTCCTTGCTCTCGACAACACGAACAAATTGATCCTCCTC
GVGDVTGF LALDNTNKLIVL

104

TCTTTCCGTGGCTCTCGTTCCATAGAGAACTGGATCGGGAATCTTAAC^
SFRGSRS IENWIGNLNFDLK

GAAATAAATGACATTTGCTCCGGCTGCAGGGGACATGACGGCTTCACTrCGTCCT
124 EINDICSGCRGHDGFTSSWR

TCTGTAGCCGATACGTTAAGGCAGAAGGTGGAGGATGCTGTGAG
144 SVADTLRQKVEDAVREHPDY

CGCGTGGTGTTTACCGGACATAG CTTGGGTGGTGCATTGGCAACTGTTGCCGGAGCAGAC
164 RVVFTGHS LGGALATVAGAD

CTGCGTGGAAATGGGTATGATATCGACGTGTTTTCATATGGCGCCCCCC
184 LRGNGYD IDVFSYGAPRVGN

AGGGCTTTTGCAGAATTCCTGACCX^ACAGACCGGCGGAACACTCTACCGCATTACCCAC
204 RAFAEFLTVQTGGTLYRITH

ACCAATGATATTGTCCCTAGACTCCCGCCGCGCGAATTCGGTTACAGCCATTCT
224 TNDIVPRLPPREFGYSHSSP

GAGTACTGGATCAAATCTGGAACCCTTGTCCCCGTCAC^
244 EYWIKSGTLV PVTRNDIVKI

GAAGGCATCGATGCCACCGGCGGCAATAACCAGCCTAACATTCCGGATATCCCTGCGCAC
264 EGIDATGGNNQPNIPDIPAH

CTATGGTACTTCGGGTTAATTGGGACATGTCTTTAG
284 LWYFGLIGTCL*

Fig. 2

WO 97/04079

PCI7DK96/00322

3/9

1
21
39
59
79
99
119
139
159
179
199
219
239
259

279

ATGAAACGCATTTGTGGTTCCCTGCTGTTGCTCGGTTTGTCGATCAGCGCCGCG
MKRICGSLLLLGLSISAALA

GAGGTCTCGCAGGATCTGTTTAACCAGTTCAATCrrCTTTGCACAGTACT
EVSQDLFNQFNLFAQYSA

GCCGCATACTGCGGAAAAAACAATGATGCCCCAGCTGGTACAAACATTACGTGCACGGGA
AAYCGKNNDAPAGTNI TCTG

AATGCCTGCCCCGAGGTAGAGAAGGCGGATGCAACGTTTCTCTAC^
NAC PEVEKADATFLYS FEDS

GGAGTGGGCGATGTCACCGGCTTCCTTGCTCTCGAC^
GVGDVTG FLALDNTNKLIVL

TCTTTCCGTGGCTCTCGTTCCATAGAGAACTGGATCG^
SFRGSRS IENWIGNLNFDLK

GAAATAAATGACATTTGCTCCGGCTGCAGGGGACATGACGGCTTCACTTCCT
EINDICSGCRGHDGFTSSWR

TCTGTAGCCGATACGTTAAGGCAGAAGGTGGAGGATGCTGTGAGGGAG
SVADTLRQKVEDAVREHPDY

CGCGTGGTGTTTACCGGACATAGCTTGGGTGGTGCATTGGC^
RVVFTGHSLGGALATVAGAD

CTGCGTGGAAATGGGTATGATATCGACGTGTTTTCATATOT
LRGNGYDIDVFSYGAPRVGN

AGGG CITTrcCAGAATTCCTGACCGTACAGACCGGCGGAACACT
RAFAEFLTVQTGGTLYR I T H

ACCAATGATATTGTCCCTAGACTCCCGCCGCGCGAATTCGGTTACAGCCAT^
TNDIVPRLPPREFGY SHSSP

GAGTACTGGATCAAATCTGGAACCCTTGTCCCCGTCACCOTAAACGA
EYWIKSGTLVPVTRNDI VKI

GAAGGCATCGATGCCACCGGCGGCAATAACCAGCCTAACATTC
EGIDATGGNNQPNIPDI PAH

CTATGGTACTTCGGGTTAATTGGGACATGTCTTTAG
LWYFGLIGTCL*

WO 97/04079

PCT/DK96/00322

WO 97/04079

PCT/DK96/00322

5/9

Eco R|, Sac I, Kpn I, Smal, BamHI

Hind IH.Sph I, Pst I
Construction of pSX578

Fig. 5

SUBSTITUTE SHEET

WO 97/04079 PCT/DK96/00322

Hind Ill.Sph I, Pst!
Construction of pSX578

Fig. 6

SI IR^sTm ITP cucrr

WO 97/04079

PCT/DK96/00322

7/9

* EcoO109

S0h I. Kfind m

pSX581

Fig. 7

WO 97/04079

PCT/DK96/00322

8/9

pJS037

Fig. 8

WO 97/04079

PCT/DK96/00322

9/9

Fig. 9

INTERNATIONAL SEARCH REPORT

International application No.

PCT/DK 96/00322

A. CLASSIFICATION OF SUBJECT MATTER

IPC6: C12N 9/20, C12N 9/18, CUD 3/386

According to International Patent Clanificaiion (IPO or to both nali

national cUtafication and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by daaificauon symbols)
IPC6: C12N

Docmnentation searched other than mmimum documentation to the extent that such documents are included in the fields searched

SE.DK.FI.NO classes as above

Electronic data base consulted during the international search (name of dau base and, where practicable.

MEDLINE, BIOSIS, EMBASE. WPI T WPIL. US PATENTS FULLTEXT. CA

search terms used)

C DOCUMENTS CONSIDERED TO BE RELEVANT

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